Biochemistry and Biophysics Reports

The current liver organ shortage has pushed the field of liver transplantation to develop new methods to prolong the preservation time of livers from the current clinical standard of static cold storage. Our approach, termed partial freezing, aims to induce a thermodynamically stable frozen state at deeper storage temperatures (-10{degrees}C to -15{degrees}C) than can be achieved with supercooling, while simultaneously maintaining a sufficient unfrozen fraction to limit dehydration and ice damage. This research first demonstrated that partially frozen glycerol treated rat livers were functionally similar after thawing from either -10 or -15{degrees}C with respect to subnormothermic machine perfusion metrics and histology. Next, we assessed the effect of adding either of two ice modulators, antifreeze glycoprotein (AFGP) and a polyvinyl alcohol/polyglycerol combination (X/Z-1000), on the viability and structural integrity of partially frozen rat livers compared to glycerol-only control livers. Results showed that AFGP livers had high levels of ATP and the least edema but suffered from significant endothelial cell damage. X/Z-1000 livers had the highest levels of ATP and energy charge (EC) but also demonstrated endothelial damage and post-thaw edema. Glycerol-only control livers exhibited the least DNA damage on Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining but also had the lowest levels of ATP and EC. Further research is necessary to optimize the ideal ice modulator cocktail for our partial-freezing protocol. Modifications to cryoprotective agent (CPA) combinations, as well as improvements to machine perfusion CPA loading and unloading, can help improve the viability of these partially frozen organs.

Huntingtons disease (HD) is a rare neurodegenerative disease. It is caused due to aggregation of Huntingtin (HTT) protein containing Q repeats more than 40. Similar protein aggregation is also a hallmark of several other neurodegenerative diseases related to loss of cognitive function. In search of modifiers of HTT aggregation, we have screened putative chaperone proteins from Drosophila in both fly and yeast model of HD. DnaJ chaperones were screened by evaluating HTT protein aggregation related phenotypes using growth assays for studying the growth rate of the cells, imaging studies, and gel-based approaches like semi-denaturing detergent agarose gel electrophoresis (SDD-AGE). Our screening led us to categorize several proteins as suppressors and enhancers of the HTT associated phenotypes. Out of the 40 chaperones and co-chaperones, two chaperones that came up strikingly were CG5001 and P58IPK. Protein aggregation was found to be reduced in both S2 cells and Drosophila transgenic lines with HTT103Q in presence of these class of chaperones. As these DnaJ chaperones have protein sequence similarity across species, these might be used as possible tools to combat the effects of neurodegenerative diseases, as evidenced specifically in Huntingtons disease and Amyotrophic Lateral Sclerosis (ALS).

In the study we showed the improvement in life longevity in mice after step-by step autologous ovarian transplantation. This has proven to be more efficient than "traditional" hormonal replacement therapy. Despite the highest speed and effectiveness of estradiol replacement deficiency in blood by its oral or transdermal use, one did not receive a significant increase in the life longevity in animals and possibly in women (Canderelli R., Leccesse L. et al. 2007). The function of transplanted fragment is usually limited to 6-12 months. This is enough for oncofertility purposes, sometimes, but not for longevity improvement. We performed periodical tissue return (autologous transplantation), containing both cortex and medulla in mice experimental model, that resulted in statistically reliable improvement of longevity. The experimental model we suggested could be projected and to other mammals or humans, as the cortical transplantation gives the same results for reproduction restoration in mice and humans and even on hormone levels normalization, but there is still lack of information about anti-ageing factors containing in ovarian medulla and cortex. That is why we consider that the most important factor for anti-ageing transplantation technology is to preserve both medulla and cortex. SummaryStep-by step autologous ovarian transplantation provides the improvement in life longevity in female mice. This technology could be even more efficient, compared to estrogen hormonal replacement therapy in hormone levels improvement (FSH, estradiol). While menopausal hormonal therapy (MHT) with estrogens doesnt improve longevity in mice, step-by-step autologous transplantation of ovarian cryopreserved tissue statistically reliably prolongs lifespan in mice.

Gain-of-function mutations in the transient receptor potential 6 (TRPC6) channel have recently been recognized as risk factors for both doxorubicin (DOX)-induced cardiomyopathy. Functional evaluation of TRPC6 missense variants is therefore important for cancer patients undergoing anthracycline treatment. However, traditional electrophysiological methods are labor-intensive and time-consuming. In this study, we compared the functional responses of TRPC6 missense variants to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC6 agonist, using molecular docking and patch clamp recording techniques. For the wild-type (WT) TRPC6 structure (PDB ID: 6UZ8), OAG exhibited a binding energy of -4.49 kcal/mol and a dissociation constant (Kd) of 0.511 mM. Twenty TRPC6 missense variants were identified from cancer patients in the Mayo Clinic database. Of these, fifteen variants had resolvable structures, nine of which displayed increased Kd values and six decreased Kd values compared to WT in molecular docking analysis. Patch clamp recordings revealed that TRPC6 WT and mutant channels were inactive at baseline but were activated upon 50 M OAG stimulation, except two loss-of-function variants. Moreover, a 24-h treatment with 0.5 M DOX significantly enhanced OAG-induced channel activation. All three variants identified in patients with heart failure demonstrated gain-of-function properties in both electrophysiological measurements and in-silico predictions. Importantly, the results obtained from molecular docking and patch clamp recordings were strongly correlated, showing an 82% concordance, higher than the predictions from AlphaMissense. These findings indicate that our computational analysis provides a rapid and reliable method for predicting the functional impact of TRPC6 missense variants, which may aid clinical decision-making in cancer patients receiving chemotherapy.

ObjectiveTo understand the effect of recombinant BSP1 (rec-BSP1) on in vitro capacitation of sperm and fertilization study Method(s)Articles were screened for reports including rec-BSP1, Capacitation, in vitro fertilization InterventionNone Main Outcome Measure(s)Reproductive outcomes, effect on gametes and embryos Result(s)Here we report an optimization of condition for rec-BSP1 production which was used for in vitro capacitation and enhancement of buffalo embryo production. The sequence of the protein was used for multiple sequence alignment which has 99% similarity with PDC 109 protein. The expression of rec-BSP1 was carried out successfully with 1 mM IPTG at 160 C for 22 hrs and purified it in soluble form. The structure of rec-BSP1 was generated using 3D modelling and analysed its mode of binding with heparin and PC by molecular docking and the structural stability of rec-BSP1-PC and rec-BSP1-heparin complexes by using molecular dynamic (MD) simulation. The effect of rec-BSP1 was observed on in vitro capacitation of spermatozoa and buffalo blastocyst production. It was found that the rec-BSP1 enhanced the sperm motility at a concentration of 50 g/ml for 1 h of incubation without having any detrimental effect on the sperm morphology and a significant increase in blastocyst production at concentration of 50 g/ml rec-BSP1. Hence this finding represents a new insight and advance the prospective approach to develop a potential fertility factor in reproduction. Conclusion(s)The purified rec-BSP1 may enhance on male fertility and mediated its effect on in vitro blastocyst production in buffalo.

Autosomal dominant polycystic kidney disease (ADPKD), a genetic disorder characterized by the formation of fluid-filled cysts within the kidneys, leading to progressive renal dysfunction, is primarily caused by mutations in PKD1, a gene encoding for the protein polycystin-1 (PC1). Understanding the structural consequences of PKD1 variants is crucial for elucidating disease mechanisms and developing targeted therapies. In this study, we analyzed the effects of nine missense PKD1 variants, including c.6928G>A p.G2310R, c.8809G>A p.E2937K, c.2899T>C p.W967R, c.6284A>G p.D2095G, c.6644G>A p.R2215Q, c.7810G>A p.D2604N, c.11249G>C p.R3750P, c.1001C>T p.T334M, and c.3101A>G p.N1034S on RNA structures, their interactions utilizing computational tools. We also explain the effects of these variants on PC1 protein dynamics, stability, and interactions using molecular dynamics (MD) simulation. These variants are located at crucial domains such as the REJ domain, PKD domains, and cation channel domain, potentially compromising PC1s function and contributing to ADPKD pathogenesis. Findings reveal substantial deviations in RNA structures and their interactions with other proteins or RNAs and also protein structure and dynamics for variants such as c.8809G>A (p.E2937K), c.11249G>C (p.R3750P), c.3101A>G (p.N1034S), c.6928G>A (p.G2310R), c.6644G>A (p.R2215Q) suggesting their potential implications in disease etiology. The study also suggests that although certain variants may have minimal effects on RNA conformations, their observed alterations in MD simulations indicate potential impact on protein structure dynamics highlighting the importance of evaluating the functional consequences of genetic variants by considering both RNA and protein levels. This study offers valuable perspectives of the utility of studying the structure dynamics through computational tools in prioritizing the variants for their functional implications and understanding the molecular mechanisms underlying ADPKD pathogenesis and developing therapeutic interventions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/586139v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@4623f0org.highwire.dtl.DTLVardef@e186a1org.highwire.dtl.DTLVardef@4deffaorg.highwire.dtl.DTLVardef@c60744_HPS_FORMAT_FIGEXP M_FIG C_FIG

HnRNP A3 is a protein that binds the age-related increase element (AIE) of blood coagulation factor IX (FIX) and that plays critical roles in age-related gene expression, likely through an epigenetic mechanism as yet unidentified. In a previous study, we found that Ser359 phosphorylated hnRNP A3 proteins do not bind to the AIE of FIX although both unphosphorylated and Ser359 phosphorylated hnRNP proteins exist in the liver. In the present study, to explore the relationship between hnRNP A3 and FIX, we examined the age-related expression pattern of 14 single spots of hnRNP A3 detected by 2DE and subsequent MALDI-TOF/TOF/MS analyses in mouse liver. We found that the level of all four Ser359 phosphorylated hnRNP A3 proteins increased with age (from 1-21 months), while the 10 unphosphorylated hnRNP A3 proteins showed various expression patterns with age. We then examined the functional role of hnRNP A3 in FIX expression using siRNA knockdown technology targeting the hnRNP A3 gene in aged mice (12-17 months old). Inhibition of hnRNP A3 expression induced an increase in the circulating FIX level in aged mice. These results suggested that hnRNP A3 inhibits age-related FIX protein expression and that age-dependent modification of hnRNP A3, including its phosphorylation at Ser359, might be involved in the age-dependent increase in FIX expression in vivo.

Cold ischemia time (CIT), the interval between tissue excision and preservation, is a critical preanalytical variable that profoundly impacts gene expression profiles. Variability in CIT can lead to inconsistent transcriptomic results, making study interpretation challenging and undermining reproducibility in biomedical research. Our study aimed to evaluate the impact of CIT on the expression of cancer-related genes, particularly these involved in hypoxia, apoptosis, and epithelial-to-mesenchymal transition (EMT). We performed RNA sequencing on 54 normal colon mucosa samples from nine patients undergoing colorectal cancer surgeries, freezing samples at predefined intervals ranging from 0 to 60 minutes. A total of 44 differentially expressed genes (DEGs) (p < 0.05) were identified when comparing samples frozen immediately (T0) with those frozen after 60 minutes (T5). These DEGs were further analyzed through functional and pathway enrichment analyses and weighted gene coexpression network analysis (WGCNA). The enrichment analysis revealed significant alterations in pathways associated with apoptosis, hypoxia, EMT, and cancer progression, including p53 and HIF-1 signaling. WGCNA highlighted two co-expressed gene modules: ME2, which showed downregulation of apoptosis-related genes, and ME4, linked to apoptosis and cellular metabolism. Our findings highlight CIT as a critical preanalytical variable, showing that prolonged ischemia can induce transcriptomic changes that may mimic malignancy, and potentially confound research outcomes. To minimize such effects, we recommend keeping CIT under 45-60 minutes.

The anticancer agent, 5-fluorouracil (5-FU), is typically applied in the treatment of various types of cancers because of its properties. Thought to be an inhibitor of the enzyme thymidylate synthase which plays a role in nucleotide synthesis, 5-FU has been found to induce single- and double-strand DNA breaks. The activation of ATR occurs as a reaction to UV- and chemotherapeutic drug-induced replication stress. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity. Using western blotting, we found that 5-FU treatment led to the phosphorylation of ATR. Surviving fractions were remarkably decreased in 5-FU with ATR inhibitor (ATRi) compared to 5-FU with other major DNA repair kinases inhibitors. ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU could induce the activation of intra-S checkpoint. Surprisingly, BRCA2-deficient cells were sensitive to 5-FU in the presence of ATRi. In addition, ATR inhibition enhanced the efficacy of 5-FU treatment, independent of non-homologous end-joining and homologous recombination repair pathways. Findings from the present study suggest ATR as a potential therapeutic target for 5-FU chemotherapy.

Postponement of fatherhood is growing worldwide due to socio-economic factors. The choice to conceive the first child above the age of 35 years is often associated with reduced fertility and poor pregnancy outcome. As widely known, several factors (e.g., lifestyle, environment, health problems) can affect spermatogenesis leading to poor reproductive outcome. Currently, the debate on the influence of aging on male gametes and safety/risk of conception at advanced age is still ongoing. Controversial results have been published so far on the changes in semen features of aging men and other mammalian species (mainly rodents). In this study, we aimed to assess how aging affects sperm quality in an inbreed mouse model, without underlying infertility, using a flow cytometry approach. Our data showed that aging is associated with increased sperm chromatin condensation, but not changes in the DNA integrity, metabolic activity or viability. These data suggest a mild effect of aging on sperm quality in a mouse model without underlying infertility.

The difficulty of cryopreservation has long been a limitation of Drosophila melanogaster as a genetic model organism. Here we report a statistically significant improvement in the efficiency of Drosophila cryopreservation by substituting limonene with the monoterpenoid fenchone in the embryo permeabilization step of a previously published method. We found that fenchone-permeabilized embryos exhibit greater uptake of cryoprotectant compared with those permeabilized by limonene, and a ~6-fold increase in the rate of egg-to-adult survival for wild-type flies. Using this improved protocol, we successfully cryopreserved and revived precious strains after 12 months of storage in liquid nitrogen. These results suggest that fenchone is a superior permeabilizing agent for fly embryo cryopreservation, expanding possibilities for the long-term maintenance of Drosophila and other insect species. Further refinement of this approach may enable cryopreservation to replace continuous culture as the method of choice for routine maintenance of fly stocks.

SLC9A6 (also termed NHE6) encodes the endosomal Na+/H+ exchanger 6 (NHE6). Pathogenic, loss-of-function mutations in NHE6 cause the X-linked neurogenetic disorder Christianson syndrome (CS). We developed induced pluripotent stem cell (iPSC) lines derived from a patient with CS and from a biologically related control. The patient with CS contained the nonsense mutation c.1569G>A (p.(W523X)), which caused a significant reduction in NHE6 mRNA and a lack of detectable NHE6 protein in CS iPSCs in comparison to control iPSCs. To establish a cell model for study of CS with an isogenic control, we corrected the c.1569G>A mutation to the NHE6 reference genome sequence using CRISPR/Cas9-mediated homology directed repair knock-in methodology. Multiple subclonal lines were generated, and notably, NHE6 protein was expressed in all analyzed c.1569G>A (p.(W523X)) genome-corrected iPSC lines. This CS iPSC model together with the associated biologically related and isogenic control cell lines will serve as a valuable resource for both basic and translational studies in CS.

The p53 protein is crucial for regulating cell survival and apoptosis in response to DNA damage. However, its influence on therapy effectiveness is controversial: when DNA damage is high p53 directs cells toward apoptosis, while under moderate genotoxic stress it saves the cells from death and promote DNA repair. Furthermore, these processes are influenced by the metabolism of transition metals, particularly copper since they serve as cofactors for critical enzymes. The metallochaperone Atox1 is under intensive study in this context because it serves as transcription factor allegedly mediating described effects of copper. Investigating the interaction between p53 and Atox1 could provide insights into tumor cell survival and potential therapeutic applications in oncology. This study explores the relationship between p53 and Atox1 in HCT116 and A549 cell lines with wild type and knockout TP53. The study found an inverse correlation between Atox1 and p53 at the transcriptional and translational levels in response to genotoxic stress. Atox1 expression decreased with increased p53 activity, while cells with inactive p53 had significantly higher levels of Atox1. Suppression of both genes increased apoptosis, while suppression of the ATOX1 gene prevented apoptosis even under the treatment with chemotherapeutic drugs. The findings suggest that Atox1 may act as one of key elements in promotion of cell cycle under DNA-damaging conditions, while p53 works as an antagonist by inhibiting Atox1. Understanding of this relationship could help identify potential targets in cell signaling pathways to enhance the effectiveness of antitumor therapy, especially in tumors with mutant or inactive p53.

Epidermal Growth Factor Receptor (EGFR) signaling is known to play essential roles in growth and development; nevertheless, overexpression and mutation of EGFR have been reported in several cancers. Non-small cell lung cancer (NSCLC), the most observed type of lung cancer, harbors the highest number of EGFR tyrosine kinase mutations and therefore, EGFR has become an important therapeutic target for treatment of these tumors. Tyrosine Kinase Inhibitors (TKIs) are found to be effective in patients whose tumors contain activating mutations in the tyrosine kinase region of the receptor. This would seem to be beneficial in the treatment of EGFR mutation-positive NSCLC patients but the activating mutations should be sensitive to TKIs. Earlier, a machine learning approach was developed to classify single amino acid polymorphisms (SAPs) in EGFR into driver (cancer-causing) and passenger (neutral) mutations using structural and functional features (Anoosha et al., 2015). This study screened all possible point mutations in EGFR and predicted a list of mutations with high probability of being a driver or a passenger. From this list, we selected 2 mutations (G729E and G719F) with high evolutionary conservation score for in vitro validation. If proven to be oncogenic drivers and sensitive to EGFR TKIs, these mutations can aid in the early diagnosis and successful therapy of EGFR mutation-positive NSCLC.

1Members of the parvalbumin (PV) family of calcium binding proteins are found in a variety of vertebrates, where can they influence neural functions, muscle contraction and immune responses. It was reported that the -parvalbumin (PV)s AB domain comprising two -helices, dramatically increases the proteins calcium (Ca2+) affinity by {approx}10 kcal/mol. To understand the structural basis of this effect, we conducted all-atom molecular dynamics (MD) simulations of WT PV and truncated -parvalbumin ({Delta}PV) constructs. Additionally, we also examined the binding of magnesium (Mg2+) to these isoforms, which is much weaker than Ca2+ (Mg2+ actually does not bind to the {Delta}PV). Our key finding is that reorganization energies (RE) assessed using molecular mechanics generalized Born approximation (MM/GBSA) correctly rank-order the variants according to their published Ca2+ and Mg2+ affinities. The [Formula] of the {Delta}PV compared to the wild-type (WT) is 415.57{+/-}0.55 kcal/mol, indicating that forming a holo state of {Delta}PV in the presence of Ca2+ incurs a greater reorganization penalty than the WT. This is consistent with the {Delta}PV exhibiting lesser Ca2+ affinity than the WT ({approx}9.5 kcal/mol). Similar trend was observed for Mg2+ bound variants as well. Further, we screened for metrics such as oxygen coordination of EF hand residues with ions and found that the total oxygen coordination number (16 vs. 12 in WT:Ca2+ and {Delta}PV:Ca2+) correlate with the reported ion affinities (-22 vs. -12.6 kcal/mol in WT:Ca2+ and {Delta}PV:Ca2+), which indicates that AB domain is required for the protein to coordinate with maximal efficiency with the binding ions. To our surprise, no significant differences were observed between the Mg2+ bound WT and {Delta}PV isoforms. Additionally, we have screened for factors such as total number of waters, hydrogen bonds, protein helicity and {beta}-content for the entire protein, which enables us to understand the impact of lack of AB domain on the entire structure and not just binding sites. Our data indicate that AB improves the overall helicity ({approx}5%) in apo as well as holo forms. Particularly, AB increases -helicity in the D-helix residues (i.e., 60-65) upon ion binding by {approx}35% (90% vs. 55% in the Ca2+ bound WT and {Delta}PV, 60% vs. 20% in the Mg2+ bound WT and {Delta}PV), which likely contributes to high Ca2+ binding affinity. On the contrary, no significant effect on the overall {beta}-content was observed. Similarly, increased dehydration ({approx}50) and increase in total number of hydrogen bonds ({approx}7) were observed upon ion binding in both the WT and {Delta}PV systems, however, no significant differences were observed between the WT and {Delta}PV variants and also between Ca2+ and Mg2+ isoforms. We speculate that this is due to the partially folded apo state that was captured in our MD simulations, which might not be physiologically relevant as suggested by NMR experiments [1]. Also, we have identified seven different interactions that might play a key role in binding the AB domain with the CDEF helices, particularly the D22(AB)-S78(CDEF) hydrogen bond. Overall, this study indicates that local (i.e., the EF hands) as well as global factors play a role in improved ion binding due to AB domain.

In fish, growth hormone (GH)-transgenesis may modify physiological mechanisms of adaptation when challenged by biotic and abiotic stressors. Thus, we evaluated whether GH overexpression can alter the thermal tolerance of adult and juvenile GH-transgenic zebrafish (Danio rerio). This study compared the thermal tolerance in non-transgenic (NT) and GH-transgenic (T) zebrafish exposed to 13 {degrees}C, 39 {degrees}C, or 28 {degrees}C (control) for 96 h. Mortality rate was checked every 12 h in juvenile (8 week-old) and adult males (6 month-old). Exposure to different temperatures revealed that GH overexpression increases the tolerance of transgenic juveniles exposed to 13 {degrees}C and diminishes the tolerance of juveniles and adults, when exposed to 39 {degrees}C. Additionally, we have analyzed transcriptional expression from the heat shock proteins (HSPs), which are mainly involved in the thermal tolerance mechanism. The mRNA level analysis results revealed that, under controlled conditions (28 {degrees}C), GH-transgenesis upregulates the expression of hsp47, hsp70, hsp90a and heat shock transcription factor (hsf1a) in transgenic juveniles, although the same result was not observed in transgenic adults. Exposure to low temperature did not alter the expression of any analyzed gene, both in adults and in juveniles. Exposure to 39 {degrees}C decreased the expression of all genes analyzed, in GH-transgenic adults. Furthermore, the HSP expression pattern was analyzed via hierarchical clustering. This analysis revealed two major clusters illustrating the dependency of gene changes related to age. These results indicate that the GH overexpression can alter thermal tolerance of fish, depending of age and temperature. Highlights- GH-transgenesis increased the survival rate of juveniles at low temperature; - High temperature is more lethal for juvenile ande adult GH-transgenic zebrafish; - GH-transgenesis increased expression of hsf1a, hsp47, and hsp70 genes in juvenile zebrafish; - hsf1a, hsp47, hsp70, hsp90a, and hsp90b genes expression is diminished in adult zebrafish GH-transgenesis exposure at high temperature. O_FIG O_LINKSMALLFIG WIDTH=142 HEIGHT=200 SRC="FIGDIR/small/445844v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1847289org.highwire.dtl.DTLVardef@7bb056org.highwire.dtl.DTLVardef@75efc9org.highwire.dtl.DTLVardef@d1a9ac_HPS_FORMAT_FIGEXP M_FIG C_FIG

The control of Hsp70 functions has been related to the modulation of ATP hydrolysis and substrate capture by Hsp40. Structural and biophysical analyses of Hsp40 variants and their interactions with Hsp70 have identified key residues for this functional control mechanism. Conserved residues in both Hsp40 and Hsp70 have revealed conserved interactions that link Hsp40 binding to the catalytic residues within Hsp70. The current work investigates the effect of documented J-domain dysfunctional mutations (i.e. D35N, H33Q) on the described interaction linkage. Molecular dynamics simulations were used to compare the persistence of individual bond types (i.e. H-bonds, salt bridges, hydrophobic interactions) between Hsp70 and the bound forms of functional and dysfunctional Hsp40 variants. The generated data suggests the involvement of both direct and allosteric effects for the tested mutations. The observed changes relate mutations in the conserved HPD tripeptide of Hsp40 to alterations in the interaction network that induces Hsp70 chaperone functions. STATEMENT OF SIGNIFICANCEThe significance of the work may be summarized as follows. First, the interaction network for the simulated systems were observed to be different from one previously proposed for a disulfide linked complex (9). This may be attributed to altered residue movement and interactions without the restrictions set by the disulfide link. These results support the use of in silico methods to refine investigations of molecular contacts, particularly for systems, whose in vitro structural elucidation are difficult to achieve without modifications. Second, key interactions for intermolecular and intramolecular contacts were observed within a short simulation time (0.1 ns) matched those from much longer runs (500 ns) (4). This result highlights the possibility of identifying key interactions with relatively low computational cost.

Ambient temperature is an important non-biotic environmental factor influencing immunological and oncological parameters in laboratory mice. It is under discussion which temperature is more appropriate and whether the commonly used room temperature in rodent facilities of about 21{degrees}C represents a chronic cold stress or the 30{degrees}C of the thermoneutral zone constitutes heat stress for the animals. In this study we selected the physiological challenging period of lactation to investigate the influence of a cage temperature of 20{degrees}C, 25{degrees}C, and 30{degrees}C, respectively, on reproductive performance and stress hormone levels in two frequently used mouse strains. We found that more pups were weaned from B6D2F1 hybrids compared to C57BL/6N mothers and that the number of weaned pups was strongly reduced if mothers of both strains were kept at 30{degrees}C. Furthermore, at 30{degrees}C mothers and pups showed reduced body weight at weaning and offspring had longer tails. Despite pronounced temperature effects on reproductive parameters, we did not find any impact on adrenocortical activity in breeding and control mice. Independent of the ambient temperature however, we found that females raising pups showed elevated levels of fecal corticosterone metabolites (FCMs) compared to controls. Increased levels of stress hormone metabolites were measured specially around birth and during the third week of lactation. Our results provide no evidence for reduced or improved wellbeing of lactating mice at different ambient temperatures, but we found that a 30{degrees}C cage temperature impairs reproductive performance.

Glioblastoma aggressively invades surrounding tissue by expressing matrix metalloproteinase-2 (MMP-2). Therefore, effective inhibition of MMP-2 is a desirable target for treatment. In some reports, the chlorotoxin (Ctx) polypeptide produced by the scorpion Leiurus quinquestriatus, interacts with human MMP-2 to inhibit tumor invasion without affecting surrounding tissue. We employed three molecular docking methodologies followed by molecular dynamics simulations to find consensus binding and calculate the binding energy of these peptide ligands to MMP-2. In addition to the Ctx itself, four C-terminal fragments were chosen to study their binding to MMP-2. The molecular docking platforms HPEPDOCK, HADDOCK, and AlphaFold2 created peptide - protein poses for each candidate binding to MMP-2. These poses underwent 500 ns molecular dynamics simulations. Peptide binding on MMP-2 and final binding energies were calculated using the Molecular Mechanics Poisson-Boltzmann Surface Area method. Configurational entropy and root-mean square deviation analyses showed stable peptide - protein complexes. Ctx and its peptide fragments frequently bound to regions on MMP-2 other than the catalytic site. All docking methods shared consensus on large negative binding energies, indicating favorable interaction between Ctx and its analogs with MMP-2. While Ctx and its fragments bind to MMP-2, there is no consensus on which region of MMP-2 they are bound to or which peptide binds strongest. Neither Ctx nor its fragments inhibited MMP-2 enzymatic activity, however, glioblastoma cellular migration was inhibited. Interactions with the non-catalytic regions of MMP-2 suggest allosteric binding to MMP-2. Inhibition of cellular migration without inhibition of MMP-2 activity warrants further study into the possible targets of Ctx expressed in glioblastoma.

Fanconi anaemia (FA) is a rare, recessive, genetic disorder characterised by a predisposition to cancer. Patients with FA are 700 times more likely to develop head and neck squamouscell carcinomas including oral epithelial dysplasia (OED), a potentially malignant disorder of the oral mucosa. This increased likelihood suggests that the molecular mechanism responsible for dysplastic transformation may involve defects in the FA pathway. In this study, the significance of ataxia telangiectasia and Rad3-related protein (ATR), which is responsible for homologous recombination repair (HRR), was investigated. ATR protects against mutations in both normal squamous cells and cancer cells by inducing HRR, and thus is associated with radiotherapy resistance. This investigation was designed to study the effects of heat on DNA repair specific to the FA pathway. Western blotting was carried out to determine whether heat affects the ATR pathway, followed by survival assays to determine the viability of cells after heat treatment and then compared to cells treated with a specific ATR inhibitor. This project aims to discover whether heat could be used as a non-invasive treatment to increase the sensitivity of tumour cells towards radiotherapy leading to an improved treatment plan for patients suffering from head and neck cancers.