Biochemical and Biophysical Research Communications

Since its outbreak in 2019 SARS-CoV-2 has spread with high transmission efficiency across the world, putting health care as well as economic systems under pressure [1, 2]. During the course of the pandemic, the originally identified SARS-CoV-2 variant has been widely replaced by various mutant versions, which showed enhanced fitness due to increased infection and transmission rates [3, 4]. In order to find an explanation, why SARS-CoV-2 and its emerging mutated versions showed enhanced transfection efficiency as compared to SARS-CoV 2002, an improved binding affinity of the spike protein to human ACE has been proposed by crystal structure analysis and was identified in cell culture models [5-7]. Kinetic analysis of the interaction of various spike protein constructs with the human ACE2 was considered to be best described by a Langmuir based 1:1 stoichiometric interaction. However, we demonstrate in this report that the SARS-CoV-2 spike protein interaction with ACE2 is best described by a two-step interaction, which is defined by an initial binding event followed by a slower secondary rate transition that enhances the stability of the complex by a factor of [~]190 with an overall KD of 0.20 nM. In addition, we show that the secondary rate transition is not only present in SARS-CoV-2 wt but is also found in B.1.1.7 where its transition rate is five-fold increased.

Vertebrates show conserved left-right (L-R) asymmetry of internal organs controlled by Nodal-Pitx2/Lefty signaling [1-3]. Modifications in L-R asymmetry occur in mutants [4] and rarely in humans [5], but little is known about natural L-R changes during evolution. Here we describe changes in L-R asymmetry in Astyanax mexicanus, a teleost with ancestral surface (surface fish) and derived cave (cavefish) morphs [6]. In teleosts, Nodal-Pitx2 signaling is activated in the left lateral plate mesoderm (LPM), the cardiac tube jogs to the left and loops to the right (D-looping), and the liver and pancreas form on opposite sides of the midline. Surface fish show conventional L-R patterning, but cavefish can show Nodal-Pitx2 expression in the right LPM or bilaterally, left (L)-looping hearts, and reversed liver and pancreas asymmetry, and these reversals have no effect on survival. The Lefty1 Nodal antagonist is expressed along the surface fish and cavefish midlines, but expression of the Lefty2 antagonist is absent in the LPM of most cavefish embryos, suggesting a role for lefty2 (lft2) in changing organ asymmetry. Although CRISPR-Cas9 lft2 editing affected D-looping in surface fish, the cavefish lft2 gene showed no coding mutations, and was expressed normally during cavefish gastrulation, suggesting downregulation by regulatory changes. Reciprocal hybridization, the fertilization of cavefish eggs with surface fish sperm and vice versa, indicated that the change in cavefish L-R asymmetry is a maternal genetic effect. Our studies reveal natural changes in internal organ asymmetry during evolution and introduce A. mexicanus as a new model to study the underlying mechanisms.

The SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus) has accumulated multiple mutations during its global circulation. Recently, a new strain of SARS-CoV-2 (VUI 202012/01) had been identified leading to sudden spike in COVID-19 cases in South-East England. The strain has accumulated 23 mutations which have been linked to its immune evasion and higher transmission capabilities. Here, we have highlighted structural-function impact of crucial mutations occurring in spike (S), ORF8 and nucleocapsid (N) protein of SARS-CoV-2. Some of these mutations might confer higher fitness to SARS-CoV-2. SummarySince initial outbreak of COVID-19 in Wuhan city of central China, its causative agent; SARS-CoV-2 virus has claimed more than 1.7 million lives out of 77 million populations and still counting. As a result of global research efforts involving public-private-partnerships, more than 0.2 million complete genome sequences have been made available through Global Initiative on Sharing All Influenza Data (GISAID). Similar to previously characterized coronaviruses (CoVs), the positive-sense single-stranded RNA SARS-CoV-2 genome codes for ORF1ab non-structural proteins (nsp(s)) followed by ten or more structural/nsps [1, 2]. The structural proteins include crucial spike (S), nucleocapsid (N), membrane (M), and envelope (E) proteins. The S protein mediates initial contacts with human hosts while the E and M proteins function in viral assembly and budding. In recent reports on evolution of SARS-CoV-2, three lineage defining non-synonymous mutations; namely D614G in S protein (Clade G), G251V in ORF3a (Clade V) and L84S in ORF 8 (Clade S) were observed [2-4]. The latest pioneering works by Plante et al and Hou et al have shown that compared to ancestral strain, the ubiquitous D614G variant (clade G) of SARS-CoV-2 exhibits efficient replication in upper respiratory tract epithelial cells and transmission, thereby conferring higher fitness [5, 6]. As per latest WHO reports on COVID-19, a new strain referred as SARS-CoV-2 VUI 202012/01 (Variant Under Investigation, year 2020, month 12, variant 01) had been identified as a part of virological and epidemiological analysis, due to sudden rise in COVID-19 detected cases in South-East England [7]. Preliminary reports from UK suggested higher transmissibility (increase by 40-70%) of this strain, escalating Ro (basic reproduction number) of virus to 1.5-1.7 [7, 8]. This apparent fast spreading variant inculcates 23 mutations; 13 non-synonymous, 6 synonymous and 4 amino acid deletions [7]. In the current scenario, where immunization programs have already commenced in nations highly affected by COVID-19, advent of this new strain variant has raised concerns worldwide on its possible role in disease severity and antibody responses. The mutations also could also have significant impact on diagnostic assays owing to S gene target failures.

The human prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-linked membrane-bound glycoprotein, containing two glycosylation sites. Human PrP is associated with a number of neurodegenerative diseases, called transmissible spongiform encephalopathies (TSE). Pathogenesis involves a structural conversion of the cellular form (PrPC), rich in -helical and random coil structure, into the scrapie form (PrPSc) characterized by parallel in register intermolecular {beta}-sheet conformation. To get a better understanding of this structural conversion, it is crucial to first characterize the non-pathogenic cellular isoform including all posttranslational modifications, like GPI-anchoring and native-like human glycosylation pattern. So far, studies on PrPC or PrPSc as well as the transition from one state to the other rely on non-native constructs of PrP studied far away from physiological conditions. We, therefore, established the expression of GPI-linked human PrP with close to native glycosylation pattern (native-like human PrP) using the eukaryotic LEXSY expression system in Leishmania tarentolae. This expression system has the added advantage that it allows for large-scale production of the native-like human PrP, which results in [~]1 mg purified protein per liter culture. Sedimentation velocity analysis and far-UV circular dichroism spectroscopy confirm the high structural homogeneity and monomeric native-like conformation of the purified GPI-anchored human PrP.

The placenta is an important producer of hormones essential for fetal development. Insulin-like growth factor 1 (IGF1) is a hormone primarily produced in the placenta in utero and is an important regulator of various developmental pathways including those in heart and liver. Embryonic disruptions in these developmental pathways can lead to lifelong changes and are often associated with chronic disease. Further, the placenta has sex-specific impacts on offspring development in response to hormonal changes. Previous work has shown that altered expression of Igf1 in the placenta results in sexually dimorphic changes to placental and fetal developmental outcomes. Here, mice underwent placental-targeted CRISPR manipulation for overexpression or insufficiency of Igf1. At the time of euthanasia, heart and liver tissues were collected and weighed. This dataset presents the heart and liver mass of these postnatal mice. There was a significant increase in proportional heart mass in placental Igf1 overexpression adult female mice and a trending increase in proportional liver mass in placental Igf1 overexpression adult male mice. No significant changes in heart or liver mass were seen in placental Igf1 insufficiency mice. These data provide insight into the impact of placental IGF1 on long-term heart and liver development. VALUE OF THE DATAO_LIThere is significant evidence for the role of early genetic changes in influencing long-term health outcomes, as laid out by the Developmental Origins of Health and Disease (DOHaD) hypothesis [1]. According to this hypothesis, genetic factors may be critical in determining the timing and severity of chronic disease, with varying effects based on sex. Genetics of the placenta, which makes up the maternal-fetal interface, plays an important role in modulating exposures associated with the DOHaD hypothesis [2]. C_LIO_LIThe placenta provides essential hormones to the fetus during pregnancy [3]. Placental changes are associated with the development of chronic disease and metabolic changes [4,5]. Disruptions in placental functions have been linked to defects including congenital heart disease which affects approximately 40,000 babies each year in the United States [6,7]. The placenta is also linked to metabolic diseases later in life such as nonalcoholic fatty liver disease, a chronic liver disease which has increased in prevalence by over 50% from 1990 to 2019 [5,8,9]. C_LIO_LIInsulin-like growth factor 1 (IGF1) is a placentally produced factor that regulates pathways involved in fetal growth and development and has been shown to be critical in growth of the heart and liver [10-13]. Despite the importance of the placenta and IGF1 in heart and liver growth, specific links between placental Igf1 expression and developmental outcomes remain understudied. C_LIO_LIPlacental function is known to have sex-specific impacts on fetal growth [14]. Further, Igf1 expression in the placenta is linked to differences in offspring developmental outcomes by sex [15]. Placental Igf1 overexpression and insufficiency affect offspring in a sexually dimorphic manner. IGF1 is a hormone and interacts with sex hormones, likely contributing to sex differences in response to changes in Igf1 expression [16]. Further research, including the work done to produce this dataset, may help clarify the role of placenta Igf1 expression in fetal outcomes, specifically regarding sex differences. C_LIO_LIThe data presented in this paper provide insight into the effects of placental Insulin-like growth factor 1 overexpression and insufficiency on adult heart and liver mass. More research is needed to understand specific functional impacts on these organs. Further, understanding the effects of placental genetic changes may support the development of future treatments and therapies for placental insufficiencies. C_LI

Since SARS-CoV-2 Omicron variant (B.1.1.529) was reported in November 2021, it has quickly spread to many countries and outcompeted the globally dominant Delta variant in several countries. The Omicron variant contains the largest number of mutations to date, with 32 mutations located at spike (S) glycoprotein, which raised great concern for its enhanced viral fitness and immune escape[1-4]. In this study, we reported the crystal structure of the receptor binding domain (RBD) of Omicron variant S glycoprotein bound to human ACE2 at a resolution of 2.6 [A]. Structural comparison, molecular dynamics simulation and binding free energy calculation collectively identified four key mutations (S477N, G496S, Q498R and N501Y) for the enhanced binding of ACE2 by the Omicron RBD compared to the WT RBD. Representative states of the WT and Omicron RBD-ACE2 systems were identified by Markov State Model, which provides a dynamic explanation for the enhanced binding of Omicron RBD. The effects of the mutations in the RBD for antibody recognition were analyzed, especially for the S371L/S373P/S375F substitutions significantly changing the local conformation of the residing loop to deactivate several class IV neutralizing antibodies.

The gastric peptide ghrelin and its receptor GHSR have important functions in energy metabolism. Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was identified as an endogenous antagonist for GHSR. Ghrelin, LEAP2, and GHSR are ubiquitously present from fishes to mammals and are highly conserved in evolution. However, our recent study suggested that GHSRs from the Actinopterygii fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost their binding to ghrelin, despite binding normally to LEAP2. Do these fish GHSRs use another peptide as their agonist? To answer this question, in the present study, we tested to two fish motilins that are closely related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the GHSR from the Sarcopterygii fish Latimeria chalumnae (coelacanth) bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in D. rerio and L. crocea, but is an efficient agonist for GHSR in L. chalumnae, which is known as a living fossil and is believed to be one of the closest fish ancestors of tetrapods. The results of present study suggested that in ancient fishes, GHSR had two efficient agonists, ghrelin and motilin; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins. Our present work shed new light on the ligand usage of GHSR in different fish species and in evolution.

Glucose has important roles in the development of hematopoietic stem cells and brain of zebrafish, the vertebrate animal model; however, in most oviparous animals, the amount of maternally provided glucose in the yolk is scarce. For these reasons, developing animals need some ways to supplement glucose. Recently, it was found that developing zebrafish, a teleost fish, undergo gluconeogenesis in the yolk syncytial layer (YSL), an extraembryonic tissue that surrounds the yolk, utilizing yolk nutrients as substrates. However, teleost YSL is evolutionarily unique, and it is not clear how other vertebrates supplement glucose. In this study, we used cloudy catshark, an elasmobranch species which possesses a YSL-like tissue during development, and sought for possible gluconeogenic activities in this tissue. In the catshark yolk sac, an increase in glucose level was found, and our isotope tracking by 13C-labeled substrate combined with LC/MS analysis detected gluconeogenic activities with glycerol most preferred substrate. In addition, expression analysis for gluconeogenic genes showed that many of these were expressed at the YSL-like tissue, suggesting that cloudy catshark engages in gluconeogenesis in this tissue. The gluconeogenesis in teleost YSL and a similar tissue in elasmobranch species implies conservation of the mechanisms of yolk metabolism between these two lineages. Future studies on other vertebrate taxa will be helpful to understand the evolutionary changes in the modes of yolk metabolism that vertebrates have experienced.

Since the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in several somatic cells, little is known about the infection of SASRS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that SARS-CoV-2 pseudovirus and GX_P2V could infect lung progenitor and even anterior foregut endoderm cells causing these cells death, which differentiated from human embryonic stem cells (hESCs). The infection and replication of SARS-CoV-2 and GX_P2V were inhibited when treated with whey protein of breastmilk and Remdesivir, confirming that these two viruses could infect lung progenitor and even anterior foregut endoderm. Moreover, we found that SARS-CoV-2 pseudovirus could infect endoderm and ectoderm. We found that whey protein blocked SARS-CoV-2 infecting these cells. In line with the SARS-CoV-2 results, GX_P2V could also infected endoderm and ectoderm, and also was inhibited by Remdesivir treatment. Although expressing coronavirus related receptor such as ACE2 and TMPRSS2, mesoderm cells are not permissive for SARS-CoV-2 and GX_P2V infection, which needed further to study the mechanisms. Interestingly, we also found that hESCs, which also express ACE2 and TMPRSS2 markers, are permissive for GX_P2V but not SARS-CoV-2 pseudovirus infection and replication, indicating the widespread cell types for GX_P2V infection. Heparin treatment blocked efficiently viral infection. These results provided insight that these stem cells maybe provided a stable repository of coronavirus function or genome. The potential consequence of SARS-CoV-2 and animal coronavirus such as GX_P2V infection in hESCs, germ layer and induced progenitors should be closely monitored.

Photoreceptor outer segments projecting from the surface of the neural retina toward the retinal pigment epithelium (RPE) are surrounded by a carbohydrate-rich matrix, the interphotoreceptor matrix (IPM) [1,2]. This extracellular compartment is necessary for physiological retinal function. However, specific roles for molecules characterizing the IPM have not been clearly defined [3]. Recent studies have found the presence of nonsense mutations in the interphotoreceptor matrix proteoglycan 2 (IMPG2) gene in patients affected by autosomal recessive Retinitis Pigmentosa (arRP) [4,5] and autosomal dominant and recessive vitelliform macular dystrophy (VMD) [6,7]. The gene encodes for a proteoglycan synthesized by photoreceptors and secreted in the IPM. However, little is known about the function and structure of this protein. We used the teleost zebrafish (D.rerio) as a model to study IMPG2 expression both during development and in adulthood, as its retina is very similar in humans [8]. In zebrafish, there are two IMPG2 proteins, IMPG2a and IMPG2b. We generated a phylogenetic tree based on IMPG2 protein sequence similarity among different vertebrate species, showing a significant similarity despite the evolutionary distance between humans and teleosts. In fact, human IMPG2 and D.rerio IMPG2a and IMPG2b share conserved SEA and EGF-like domains. Homology models of these domains were obtained by using the iTasser server. Finally, expression analyses of impg2a and impg2b during development and in the adult fish showed expression of both mRNAs starting from 3 days post fertilization (dpf) in the outer nuclear layer of zebrafish retina that continues throughout adulthood. This data lays the groundwork for the generation of novel and most needed animal models for the study of IMPG2-related inherited retinal dystrophies.

BackgroundCa2+-induced Ca2+ release (CICR) has been the prevailing model in cardiomyocytes for over 50 years. However, whether Na+ influx plays a direct role in triggering Ca2+ release has remained unclear. We previously discovered that action potential (AP) phase 0 triggered Ca2+ release while phase 2 regulated the decay phase during the Ca2+ transient (CT). We hypothesized that CICR might only exist under experimental conditions, not during physiological AP cycles. MethodsSimultaneous recordings of ion currents and CT from the same rat left ventricular cardiomyocyte were employed to investigate the temporal relationship between cation influxes and CT. ResultsThe reactivation time for CT was approximately 250 ms. AP phase 0 triggered the CT, while phase 2 only regulated CT width. Pulses from -40 to +80 mV with 6 ms duration were applied to validate whether the reverse Na+/Ca2+ exchanger (NCX) could trigger CT. Pulses of -40 and -30 mV with 6 ms duration induced giant Na+ influx and tail current, but only Na+ influx triggered CT, not tail current. Pulses from -20 mV to +80 mV with 6 ms duration triggered CT via tail current, not via reduced Na+ influx. In Ca2+ free solution, Na+ influx was still able to trigger CT until sarcoplasmic reticulum (SR) Ca2+ content was remarkably reduced. Moreover, increased reverse NCX current induced by ramp-like depolarizing pulses did not trigger CT but widened its shape. Additionally, when SR function was destroyed by caffeine (20 mM/L), field stimulations caused progressively elevated cytosolic Ca2+ levels, indicating that SR has a buffering function for maintaining cytosolic Ca2 concentration. ConclusionsOnce Na+ influx triggers CT, Ca2+ influx cannot trigger a second CT during an AP cycle due to the limitation imposed by the reactivation time. Na+ influx might enter SR via an unknown channel or transporter at the terminal cisternae during the AP and induce Ca2+ release by acting on the SR luminal-facing site of the ryanodine receptor 2 (RyR2) channel and/or by increasing the positive potential gradient across the SR membrane.

ABSTRACTGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited enzymopathy in humans and is associated with a predisposition to hemolysis. However, there are few animal models to that adequately mimic associated human disease states that could be used to evaluate strategies to address clinical syndromes attributable to G6PD deficiency. In the present study, we aimed to establish a stable transgenic zebrafish model of G6PD deficiency that recapitulates the clinical manifestations of G6PD deficiency. We incorporated a stable transgene of G6PD lacking nucleotides from 1315 to 1443 denoted Tg(zgata1:g6pdM1315-1443-egfp). Functional analysis showed that Tg(zgata1:g6pdM1315-1443-egfp) transgenic zebrafish demonstrate a decrease in g6pd activity, reduced GSH levels and hemoglobin content, and increases in pericardial edema in response to α-naphthol exposure, similar to human subjects with G6PD deficiency. We detected no other significant phenotypic abnormalities compared to controls. Taken together, these observations indicate that the Tg(zgata1:g6pdM1315-1443-egfp) zebrafish line mirrors key clinical manifestations of G6PD deficiency in humans. This model may facilitate mechanistic studies and promote translational research related to G6PD deficiency.Competing Interest StatementThe authors have declared no competing interest.View Full Text

The MYC associated factor X (MAX) is the heterodimeric partner of the MYC paralogs (MYC, MYCN and MYCL). When deregulated, high level of the MYC paralogs contribute to all aspects of tumorigenesis and tumor growth. MAX can also heterodimerize with the MXD proteins, MNT and MGA. Heterodimerization and sequence specific DNA binding to the E-Box sequences at gene promoters is controlled by their heterodimerization with the MAX b-HLH-LZ. As a heterodimer with MAX, MYC proteins activate genes involved in cell metabolism, growth and proliferation whereas MXD proteins, MNT and MGA repress them. MAX can also bind to the E-Bos sequence as a homodimer. Being devoid of a transactivation domain it can act as an antagonist of the MYC/MAX heterodimers. Variants of MAX have been reported to be linked to cancer. These variants are either not expressed, inactivated or lead to missense mutations. This has led to the notion that MAX may have a tumor suppressor role. Here, we characterize three of those variants with missense mutations in the basic region, i.e. E32K, R35P and R35C. We analyzed their heterodimerization with the b-HLH-LZ of MYC and their DNA binding properties as homo-and heterodimers. The R35C variant b-HLH-LZ was found to have a markedly increased affinity for the b-HLH-LZ of MYC. We also observed that all three b-HLH-LZ variants have a lower affinity as homodimers for the E-Box than the WT. This was shown to lead to a preferential binding of all the heterodimeric b-LHLH-LZ to the E-Box. This effect is exacerbated in the case of the R35C variant. We argue that this preferential binding of MYC as heterodimers with these variants to E-Box sequences could contribute to tumorigenesis. Hence, our results suggest that, mechanistically, the MAX homodimer bound to the E-Box could act as a tumor suppressor. MATERIALS AND METHODSO_ST_ABSMolecular modelingC_ST_ABSThe open source version 1.7.6.0 of Pymol was used for modeling and molecular rendering [1]. The crystal structure of the MAX homodimer bound to the E-Box (1HLO [2]) was used as a template for the generation of the models. The variants were generated using the mutagenesis function in the wizard. The conformation of the K32 side chain was manually set in order to avoid introducing steric clashes with DNA. Protein expression and purificationThe cDNA, coding for the MAX b-HLH-LZ (Max* hereafter, residues 22-103, UniProt entry P61244-1) to which are added the GSGC residues in c-terminal, inserted in the pET3a vector was already available in the laboratory [3] and was used as a template to generate the plasmids with inserts coding for each of the mutants (E32K, R35C and R35P) through quick-change PCR with Q5 DNA polymerase and DpnI from New England Biolabs. The primers used were purchased from IDT DNA, their sequences are listed in Table S1. Sequence for each construct was confirmed by Sanger sequencing at the Plateforme de sequencage SANGER - Centre de recherche du CHU de Quebec - Universite Laval. The primary structure for the basic region of each construct is given in Fig. 2A. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/715400v1_fig2.gif" ALT="Figure 2"> View larger version (41K): org.highwire.dtl.DTLVardef@1b05d5eorg.highwire.dtl.DTLVardef@1c1d692org.highwire.dtl.DTLVardef@ee469dorg.highwire.dtl.DTLVardef@15e0ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Structure schematics, specific and non-specific interactions dictating specificity and stability of binding of the basic region of MAX to the canonical (CACGTG) E-Box. A. Primary structure for the basic region of MAX and each of the variants. Positions making the most important contacts with the E-box are indicated by black arrows. Positions for the variants studied here are colored according to the Zappo colour scheme, following their physico-chemical properties: red for negative, blue for positive, magenta for proline and yellow for cysteine. B. The side chain (carboxylate) of E32 receives H-Bonds from the CA nucleobases in the leading strand (white carbon atoms). R35 and R36 make a salt bridges with phosphate groups while and the guanidino moiety of R36 makes a specific H-Bond with the nucleobase of the G in the strand of the reverse complement (cyan carbon atoms). C. The R35C mutation removes one non-specific salt-bridge at the interface of the complex. D. The aliphatic portion of the K side chain in the E32K variant is unable to accept the H-Bonds from the CA nucleobases and leads to the stabilisation of the complex and the helical structure of the basic region. E. In addition to removing a salt-bride, the Pro residue in the R35P kinks the path of the basic region, prevents the establishment of the specific H-Bonds mandatory for recognition of the E-Box and leads to unfolding of the helical state. C_FIG The MYC b-HLH-LZ (Myc*), the Max*WT b-HLH-LZ and its variants were expressed and purified as previously described [3,4] After lyophilisation, the b-HLH-LZs were kept at -20{degrees}C and solubilised in Myc buffer (50 mM NaCl, 50 mM NaH2PO4 pH 5.5) for Myc* or PBS for Max* at a final concentration of 1 mM before use. Circular dichroismAll circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier-type thermostat. The instrument was routinely calibrated using an aqueous solution of d-10-(+)-camphorsulfonic acid at 290.5 nm. Samples were prepared as follows: Max* (either WT or a variant) was diluted in 100 {micro}l 2X CD buffer (40 mM KCl, 11.4 mM K2HPO4, 28.6 mM KH2PO4, pH 6.8) and the volume adjusted to 106 {micro}l with PBS. 10 {micro}l TCEP 16 mM were added, and the volume further adjusted to 192 {micro}l with ddH2O before samples were incubated overnight at room temperature. After reduction, Myc* was added and the volume adjusted to 198 {micro}l with Myc buffer (Na2HPO4 0.95 mM, NaH2PO4 49.05 mM, 50 mM NaCl, pH 5.5). The DNA complexes were prepared as follows. After a 10 minutes incubation of the protein samples at room temperature, 0, 1 or 2 {micro}l of 2 mM of specific or non-specific DNA duplexes in 10 mM Tris pH 8.0 were added and the volume adjusted to 200 {micro}l with 10 mM Tris pH 8.0. The strands of the specific probe were: 5-ATT ACC CAC GTG TCC T*AC-3 and 5-GTA GGA CAC GTG GGT* AAT-3 (with the E-box sequence underlined) and the non-specific probe: 5-ATT ACC TCC GGA TCC T*AC-3 and 5-GTA GGA TCC GGA GGT* AAT-3 (Integrated DNA Technologies). Samples were further incubated for 10 minutes at room temperature and transferred to a 1 mm path length quartz cuvette. All spectra were recorded from 250 to 195 nm at 0.1 nm intervals by accumulating 10 spectra at 25 {degrees}C. Thermal denaturations were recorded at 222 nm from 5 to 95 {degrees}C at a heating rate of 1 {degrees}C/min. CD signal for spectra and thermal denaturations was corrected by substracting the signal from corresponding spectra or thermal denaturation either for buffer alone or the appropriate DNA duplex. CD signal was then converted to mean residue ellipticity using the following formula [5]: [{theta}] = {delta} {middle dot} MRW/(10{middle dot}c l) where [{theta}] is the mean residue ellipticity in deg {middle dot} cm2 dmol-1, {delta} is the CD signal in millidegrees, MRW is the mean residue weight, c is the concentration in mg/ml and l is the pathlength in mm. For the heterodimers, the concentration used was the sum of Max* and Myc* and the MRW was determined using a weighted average.

As intracellular organelles in adipose tissue, lipids droplets manage the balance between triglyceride accumulation and energy consumption in animals. Perilipin family members, associated with surface of lipid droplets, participate the regulation of lipid metabolism. Lipid storage droplet-1 (LSD1)/Perilipin-1 acts as a gatekeeper for adipose lipid storage in animals. Despite extensive studies in fruit fly, the function of LSD1 in insect larval stage remain indistinct. In this study, we characterized the function of LSD1 in black soldier fly Hermetia illucens, a nova resource insect to recycle organic wastes. We found that LSD1 was broadly present in dipteran species and evolved with divergence between mosquitos and flies. We further constructed in vivo mutagenesis mediated by CRISPR/Cas9 and found that mutations in LSD1 increased the larval weight and did not bring any defects in development. Raw fat content was also not significantly influenced in late larval stage and new-emerged adults. Our results not only extend our knowledge of LSD1 in insects, but also help for better understanding of the lipid homeostasis in BSF.

Circadian rhythm by Cyanobacteria is one of the simplest biological clocks: the clock consists of only three proteins, KaiA, KaiB and KaiC. Their oligomers, KaiA dimer (A2), KaiB tetramer (B4) and KaiC hexamer (C6) oscillate an association- disassociation cycle with 24hr period. In a widely accepted model, the oscillation process is as follows. From the viewpoint of a base unit (C6), C6 homo-oligomer [->] A2C6 complex [->] B6C6 complex [->] AnB6C6 complex (n[≤]12) [->]C6 homo-oligomer. In this study, Small-Angle X-ray Scattering, Contrast Matching-Small-Angle Neutron Scattering, Analytical Ultracentrifuge and phosphorylation-analysis PAGE measurements were performed to reveal the kinetics not only of KaiC hexamer but also of all components in a working Kai clock. The complementary analysis disclosed that the oscillation is not the single process as the widely accepted model but composed with synchronized multiple association-dissociation reactions between components. Namely, there are various reactions between components, which proceed simultaneously, in a working Kai-clock.

Certain viviparous animals possess mechanisms for mother-to-embryo nutrient transport during gestation. Xenotoca eiseni is one such viviparous teleost species in which the mother supplies proteins and other components to the offspring developing in the ovary. The embryo possesses trophotenia, a hindgut-derived pseudoplacenta to receive the maternal supplement. However, the molecular mechanisms underlying viviparous non-mammalian animals remain elusive. We conducted this study to investigate the mechanism for nutrient absorption and degradation in trophoenia of X. eiseni. The tracer assay indicated that a lipid transfer protein, vitellogenin (Vtg), was absorbed into the epithelial layer cells of trophotaenia. Vtg uptake was significantly suppressed by Pitstop-2, an inhibitor of clathrin-mediated endocytosis. Gene expression analysis indicated that the genes involved in endocytosis-mediated lipolysis and lysosomal cholesterol transport were expressed in trophotaenia. In contrast, plasma membrane transporters expressed in the intestinal tract were not functional in trophotaenia. Our results suggested that endocytosis-mediated lysosomal lipolysis is one of the mechanisms underlying maternal component metabolism. Thus, our study demonstrated how viviparous teleost species have acquired a unique developmental system that is based on the hindgut-derived pseudoplacenta.

Body color plays key roles in fitness, communication with others, and hiding. In poikilothermal vertebrates, the body color is mainly determined by types and distributions of chromatophores. Among them, carotenoid color of xanthophores/erythrophores is important for interspecific diversity and colorful males as sexual dimorphism. Most vertebrates cannot synthesize carotenoids in their bodies and must ingest them from food. However, the genes involved in the uptake process are not fully understood. Therefore, we tried to identify the causal gene of the carotenoid color mutant of medaka. The HdrR-II1 strain used in the genome project has orange body color in males and white body color in females. The orange and white body color was known to be controlled by the sex-linked R locus, but the causal gene of this was unknown. In this study, we identified that the causal gene of the R locus is tetratricopeptide repeat domain 39b like (ttc39bl). In the HdrR-II1, the ttc39bl on the Y chromosome is normal, but the ttc39bl on the X chromosome has an 821 bases insertion in exon 3 and is broken. This insertion is also present on both the X and Y chromosomes of commercially available white medaka.

Triacylglycerols (TAGs) are the major component of plant storage lipids. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and responsible for plant oil accumulation. We previously found DGAT activity of Vernonia galamensis DGAT1 was distinctively higher than that of Arabidopsis thaliana DGAT1 and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor were introduced into Arabidopsis (ecotype Col-0). All Vernonia DGAT1 expressing lines showed a significantly higher oil content (average 49% relative increase compared to the wild type) followed by soybean, and castor. Most Arabidopsis DGAT1 over-expressing lines did not show a significant increase. In addition to these four DGAT1s, sunflower, Jatropha and sesame DGAT1 genes were introduced into the TAG biosynthesis defective yeast mutant (H1246). In the yeast expression culture, DGAT1s from Arabidopsis, castor, and soybean only slightly increased TAG content, however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame remarkably increased TAG content more than 10 times higher than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions by site-directed mutagenesis was performed and analyzed. These substitutions substantially increased the TAG content. HighlightDGAT1s from several plant species were tested their TAG accumulation promotion in Arabidopsis and yeast. They were divided into high and low function and single amino acid substitution enhanced function

Thirty percent of all mutations causing human diseases generate mRNAs with premature termination codons (PTCs). Recognition and degradation of these PTC-containing mRNAs is carried out by the mechanism known as nonsense-mediated mRNA decay (NMD). Upf2 is a scaffold protein known to be a central component of the NMD surveillance pathway. It harbors three middle domain of eukaryotic initiation factor 4G (mIF4G) domains in its N-terminal potentially important in regulating the surveillance pathway. In this study, we defined regions within the mIF4G-1 and mIF4G-2 that are required for proper function of NMD and translation termination in Saccharomyces cerevisiae Upf2. In addition, we narrowed down the activity of these regions to an aspartic acid (D59) in mIF4G-1 which is important for NMD activity and translation termination accuracy. Taken together, these studies suggest that inherent charged residues within mIF4G-1 of Upf2 play a role in the regulation of the NMD surveillance mechanism in S. cerevisiae.

Machine learning-based platforms are currently revolutionizing many fields of molecular biology including structure prediction for monomers or complexes, predicting the consequences of mutations, or predicting the functions of proteins. However, these platforms use training sets based on currently available knowledge and, in essence, are not built to discover novelty. Hence, claims of discovering novel functions for protein families using artificial intelligence should be carefully dissected, as the dangers of overpredictions are real as we show in a detailed analysis of the prediction made by Kim et al 1 on the function of the YciO protein in the model organism Escherichia coli.