Clinical and Translational Medicine

Immunomodulatory agents dexamethasone and colchicine, antiviral drugs remdesivir, favipiravir and ribavirin, as well as antimalarial drugs chloroquine phosphate and hydroxychloroquine are currently used in the combat against COVID-191-16. However, whether some of these drugs have clinical efficacy for COVID-19 is under debate. Moreover, these drugs are applied in COVID-19 patients with little knowledge of genetic biomarkers, which will hurt patient outcome. To answer these questions, we designed a screen approach that could employ genome-wide sgRNA libraries to systematically uncover genes crucial for these drugs action. Here we present our findings, including genes crucial for the import, export, metabolic activation and inactivation of remdesivir, as well as genes that regulate colchicine and dexamethasones immunosuppressive effects. Our findings provide preliminary information for developing urgently needed genetic biomarkers for these drugs. Such biomarkers will help better interpret COVID-19 clinical trial data and point to how to stratify COVID-19 patients for proper treatment with these drugs.

Matrix metalloproteinases (MMPs) are rapidly expressed and activated in response to oxidative stress and contribute to various pathological conditions. Cisplatin is a highly effective chemotherapeutic agent; however, its clinical use is limited by its associated permanent hearing loss (ototoxicity). While cispwlatin-induced oxidative stress and inner ear cell death are well-established, the contribution of MMPs remains unclear. In this study, we demonstrate that cisplatin exposure triggers activation of MMP-2 and MMP-9 and expression of an intracellular N-terminal-truncated isoform of MMP-2 in mouse inner ear hair cells. Pharmacological inhibition of MMP-2 and genetic knockdown of Mmp-9 enhanced hair cell survival and attenuated cisplatin-induced inflammation and cytotoxicity. Furthermore, proteomic analysis revealed that proteins involved in intracellular trafficking, including RAB proteins, may serve as potential substrates of intracellular MMP-2 upon cisplatin exposure, pointing to a previously unrecognized mechanism of cisplatin-induced hair cell injury. In vitro analysis confirmed that MMP-2 cleaves RAB9A in response to cisplatin, and in silico analyses predicted MMP-2-preferred cleavage sites on RAB9A. Collectively, our findings identify MMP-2 as a promising therapeutic target for mitigating cisplatin-induced ototoxicity.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO), account for [~]25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (1) show advanced retinal degeneration in a male patient (60-70 year old) harboring four transcriptionally active intact copies of rhodopsin, (2) recapitulated the clinical phenotypes using retinal organoids, and (3) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (qRT-PCR and bulk RNA-sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300-days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that adRP due to RHO-CNV develops due protein overexpression overloading the photoreceptor post-translational modification machinery.

Cisplatin is a DNA targeting anticancer drug, yet its damaged gene loci have remained unclear. In the present work, combining affinity isolation and high throughput sequencing, we genome-widely mapped 17729 gene loci containing platination lesions, which mainly function as enzymes, transcription regulators, transporters and kinases, and of which 445 genes account for 71% of potential gene targets for cancer therapy reported in the literature. The most related core signaling pathway, disease and tissue toxicity of 7578 genes with an enrichment fold (EFG) of >12, where EFG refers to the ratio of total read counts of a gene detected in cells with and without cisplatin treatment, are sperm motility, cancer and hepatotoxicity with association P values of < 1x10-22. Among 616 kinase genes damaged by cisplatin, 427 are protein kinases which account for 82% of putative protein kinases, suggesting that cisplatin may act as broad-spectrum protein kinase inhibitor. Western Blot assays verified that expression of 8 important protein kinase genes was significantly reduced due to cisplatin damage. SPAG9 is closely related to 147 of 361 cancer diseases which the cisplatin damaged genes are associated with and was severely damaged by cisplatin. Given SPAG9 abundantly expresses JIP-4, a upstream mediator of protein kinase signaling, in testis, it may be responsible for the high sensitivity of testicular cancer to cisplatin, thus being a potential therapeutic target for precise treatment of testicular cancer. These findings provide novel insights into better understanding in molecular mechanism of anticancer activity and toxicity of cisplatin, more importantly inspire further studies in prioritizing gene targets for precise treatment of cancers.

LEAF-4L6715 is a liposomal formulation encapsulating transcrocetin (TC) developed to enhance the diffusion of oxygen in the body. Here, we report the final results of the phase I/II clinical trial (NCT04378920; EUDRACT2020-001393-30) initiated to identify an optimal regimen and to assess the activity of TC in the context of acute respiratory distress syndrome (ARDS). More specifically, LEAF-4L6715 was developed to treat patients with ARDS due to severe SARS-CoV-2 infection who have a ratio of partial arterial pressure to inspired fraction of oxygen (PaO2/FiO2 ratio) <200 treated with artificial ventilation support in an intensive care unit. A total of 37 patients were treated (across 6 dosing cohorts) with LEAF-4L6715 given as an intravenous infusion for over 90 minutes. The dose of LEAF-4L6715 was increased until the transaminase levels were elevated and 4 grade 3 events occurred among 8 patients. The recommended dosage was determined to be a fixed concentration of 300 mg administered every 12 hours. An improvement in the PaO2/FiO2 ratio and SOFA score was observed. The overall 28-day survival rate of 81%. This study identified the recommended dose for LEAF 4L6715 and the dose-limiting toxicity and showed an overall favorable risk/benefit profile. These preliminary findings are promising for the activity of LEAF-4L6715 but will require confirmation in a randomized phase III trial.

Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.

Systemic inflammation in critically ill patients can lead to serious consequences such as acute respiratory distress syndrome (ARDS), a condition characterized by the presence of lung inflammation, edema, and impaired gas exchange, associated with poor survival. Understanding molecular pathobiology is essential to improve critical care of these patients. To this end, we use multimodal profiles of SARS-CoV-2 infected hospitalized participants to the Biobanque Quebecoise de la COVID-19 (BQC-19) to characterize endophenotypes associated with different degrees of disease severity. Proteomic, metabolomic, and genomic characterization supported a role for neutrophil-associated procoagulant activity in severe COVID-19 ARDS that is inversely correlated with sphinghosine-1 phosphate plasma levels. Fibroblast Growth Factor Receptor (FGFR) and SH2-containing transforming protein 4 (SHC4) signaling were identified as molecular features associated with endophenotype 6 (EP6). Mechanical ventilation in EP6 was associated with alterations in lipoprotein metabolism. These findings help define the molecular mechanisms related to specific severe outcomes, that can be used to identify early unfavorable clinical trajectories and treatable traits to improve the survival of critically ill patients.

Long-COVID-19 manifests as a multisystemic condition with varied symptoms lingering beyond three weeks of acute SARS-CoV-2 infection, though its underlying mechanisms remain elusive. Aiming to decipher the long-term molecular impacts of COVID-19, we conducted a transcriptomic analysis on PBMCs from 1-year post-covid patients, including individuals without pneumonia (NP, n=10), those with severe pneumonia (SP, n=11), and healthy controls (C, n=13). Our extensive RNA sequencing revealed 4843 differentially expressed genes (DEGs) and 1056 differentially expressed long non-coding RNAs (DElncRNAs) in "C vs NP," 1651 DEGs and 577 DElncRNAs in "C vs SP," 954 DEGs and 148 DElncRNAs in "NP vs SP," with 291 DEGs and 70 DElncRNAs shared across all groups. We identified 14 hub genes from 291 DEGs, with functional enrichment analysis showing upregulated DEGs mainly linked to inflammation and osteoclast differentiation, and downregulated DEGs to viral infections and immune responses. These hub genes play central roles in inflammatory and immune processes and are significantly associated with pneumonitis and diverse lung diseases. Investigations revealed unique immune cell signatures across DEG categories, associating upregulated DEGs with neutrophils and monocytes, and downregulated DEGs with CD4 memory effector T cells. Analysis of 14 hub genes showed notable upregulation in the no pneumonia group versus healthy controls, displaying complex patterns in the severe pneumonia group. Our study uncovered potential idiopathic pulmonary fibrosis signals in Long-COVID-19 patients PBMC transcriptome, highlighting the urgency for thorough monitoring and extended research to understand COVID-19s lasting effects. This study sheds light on COVID-19s transcriptomic changes and potential lasting effects, guiding future research and therapeutic approaches for Long-COVID-19.

Genetic association studies have demonstrated that partial loss of SLC30A8 function protects against type 2 diabetes (T2D) in humans, but the impact of complete loss of SLC30A8 function remains unknown. From whole-exome and genome sequencing of 100,814 participants in the Pakistan Genome Resource, we identified fifteen SLC30A8 knockouts, including homozygotes for a variant enriched in South Asians (Gln174Ter) and 615 heterozygotes for loss-of-function (LoF) variants. T2D risk was lower in SLC30A8 LoF hetero- and homozygotes, and the protective effect strengthens in a gene dose-dependent manner (ORadditive=0.63 [0.53-0.78, p=7.5E-07], ORrecessive=0.27 [0.09-0.80, p=0.018]). Recall-by-genotype of SLC30A8 LoF hetero- and homozygotes and their family members with oral glucose tolerance tests showed a gene dose-dependent reduction in glucose levels coupled with elevated insulin. Corrected Insulin Response, Disposition Index, and Insulin Sensitivity Index in LoF hetero- and homozygotes indicated higher glucose-stimulated insulin secretion with preserved beta cell function. These data suggest that therapeutic knockdown of SLC30A8, up to and including complete knockout, may treat T2D safely and effectively.

Immune checkpoint inhibitors (ICIs), especially CTLA-4 inhibitors (CTLA-4), exhibit a high incidence of colitis as an immune-related adverse event (irAE) during cancer treatment, severely limiting patient benefit. Clinically, both treatment interruption and existing intervention drugs for ICI-mediated colitis may compromise antitumor efficacy. However, there is inadequate research on the pathogenesis of ICI-mediated colitis, with findings often conflicting. Here, we first established multiple clinically relevant animal models, including an immuno-humanized ICI-mediated colitis model. Through time-series transcriptomics, we discovered that CTLA-4-induced colonic toxicity exhibits characteristics ranging from early metabolic reprogramming represented by glycolysis to later immune disorders represented by Th17 responses. By targeting colonic CTLA-4+ T cells, CTLA-4 blocked CD80/CD86-CTLA-4 interaction, thereby activating the PI3K-AKT-mTOR pathway. Subsequently, mTOR mediated metabolic reprogramming in T cells, shifting them from Treg-biased oxidative phosphorylation to Th17-biased glycolysis. The colonic toxicity of CTLA-4 has also been demonstrated to depend on the PI3K-AKT-mTOR pathway, glycolysis, and Th17 responses. Notably, metformin significantly relieved ICI-mediated colitis by inhibiting mTOR without impeding antitumor efficacy. Collectively, these findings highlighted the metabolic-immune axis in the colonic toxicity of ICI and provided a clinically superior intervention strategy.

SKP1-CUL1-F-box protein (SCF) ubiquitin ligases are versatile protein complexes that mediate the ubiquitination of substrates, which are recognized by their F-box-domain- containing subunits1. One of these substrate receptors is FBXO38. Its gene has been found to be mutated in several families with early-onset distal hereditary motor neuronopathy2. SCFFBXO38 ubiquitin ligase controls the stability of ZXDB, a nuclear factor associated with the centromeric chromatin protein CENP-B3. Moreover, the loss of FBXO38 results in growth retardation and defect in spermatogenesis characterized by deregulation of the Sertoli cell transcription program and centromere integrity4. A report by Meng et al. proposed that SCFFBXO38 regulates the protein levels of the PD-1 inhibitory receptor (also known as CD279, PDCD1) in T cells5. Here, we have re-addressed the conclusions by Meng et al. using Fbxo38KO/KO mice and cell systems. We have found no evidence indicating that FBXO38 controls the abundance and stability of PD-1.

Guided by the principle of primum non nocere (first do no harm), we report a cautionary note on the potential fatal toxicity of chloroquine (CQ) or hydroxychloroquine (HCQ) in combination with anti-diabetic drug metformin. We observed that the combination of CQ or HCQ and metformin, which were used in our studies as potential anti-cancer drugs, killed 30-40% of mice. While our observations in mice may not translate to toxicity in humans, the reports that CQ or HCQ has anti-COVID-19 activity, the use of CQ resulting in toxicity and at least one death, and the recent Emergency Use Authorization (EUA) for CQ and HCQ by the US Food and Drug Administration (FDA) prompted our report. Here we report the lethality of CQ or HCQ in combination with metformin as a warning of its potential serious clinical toxicity. We hope that our report will be helpful to stimulate pharmacovigilance and monitoring of adverse drug reactions with the use of CQ or HCQ, particularly in combination with metformin.

To explore whether the expression levels of viral-entry associated genes might contribute to the milder symptoms in children, we analyzed the expression of these genes in both children and adults lung tissues by single cell RNA sequencing (scRNA-seq) and immunohistochemistry (IHC). Both scRNA-seq and IHC analyses showed comparable levels of the key genes for SARS-CoV-2 entry in children and adults, including ACE2, TMPRSS2 and FURIN, suggesting that instead of lower virus intrusion rate, other factors are more likely to be the key reasons for the milder symptoms of SARS-CoV-2 infected children.

Diabetes is associated with an increased risk of Coronavirus disease 2019 (COVID-19) vulnerability and mortality. COVID-19 vaccines significantly reduce the risks of serious COVID-19 outcomes, but the impact of COVID-19 vaccines including their effectiveness and adverse effects in patients with diabetes are not well known yet. Here, we showed that 61.1% patients with type 2 diabetes, but not healthy controls, exhibited aggravated insulin resistance towards the booster shots of the COVID-19 vaccine. Furthermore, we showed that COVID-19 vaccination once a week also impaired insulin sensitivity in healthy mice after four weeks. We further showed that metformin, a common anti-diabetic medication, improved the impaired insulin signaling induced by COVID-19 vaccination in mice. This study suggests clinical implications for the close monitoring of glycemic control in diabetic patients after receiving COVID-19 vaccines and indicates the beneficial action of metformin in counteracting insulin signaling variations induced by COVID-19 vaccination in diabetic patients.

Withdrawal StatementThe authors have withdrawn their manuscript owing to an error identified by the corresponding author. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

The contribution of the gut microbiota to distal symmetric polyneuropathy (DSPN) in diabetic patients remains elusive. We found that the gut microbiota from DSPN patients induced more severe peripheral neuropathy in db/db mice. Gut microbiota from healthy donors significantly alleviated DSPN independent from glycaemic control in patients in a randomized, double-blind and placebo-controlled trial. The gut bacterial genomes correlated with Toronto Clinical Scoring System score were organized in two competing guilds. Increased Guild 1 that had higher capacity in butyrate production and decreased Guild 2 that harbored more genes in synthetic pathway of endotoxin were associated with improved gut barrier integrity and decreased proinflammatory cytokine levels. Moreover, matched enterotype between transplants and recipients showed better therapeutic efficacy with more enriched Guild 1 and suppressed Guild 2. Thus, the two competing guilds may mediate the causative role of the gut microbiota in DSPN and have the potential to be an effective target for treatment.

BackgroundMudan granules has been used in China to treat diabetic peripheral neuropathy (DPN), but there is a lack of systematic review of reports in this area. The aim of this systematic review was to evaluate the efficacy and safety of Mudan granules in the treatment of diabetic peripheral neuropathy. MethodsInitial studies were searched from PubMed, Embase, Cochrane, China National Knowledge Infrastructure (CNKI), VIP database, Wanfang electronic databases. The Cochrane Risk of Bias tool was used to evaluate the risk of bias. The meta-analysis was performed by Stata 16.0 software. For dichotomous and continuous outcomes, the relative risk (RR) and standardized mean difference (SMD) with 95% confidence interval (CI) were conducted, respectively. Results51 randomized controlled trials (RCTs) involving 5,416 patients were included. In the meta-analysis, compared with routine treatment (RT) alone, Mudan granules plus RT reduced Toronto clinical scoring system (TCSS) score (SMD, -0.52; 95% CI, -0.66 to -0.38; P < 0.01), total symptoms score (TSS) (SMD, -1.44; 95% CI, -2.88 to -0.00; P = 0.05), serum homocysteine (Hcy) levels (SMD, - 3.84; 95% CI, -5.99 to -1.70; P < 0.01), serum high sensitive C-reactive protein (hs-CRP) levels (SMD, -1.68; 95% CI, -3.29 to -0.08; P = 0.04), and improved total clinical efficacy (RR, 1.23; 95% CI, 1.19 to 1.27; P < 0.01) and serum superoxide dismutase (SOD) levels (SMD, 1.54; 95% CI, 1.13 to 1.95; P < 0.01). Besides, Mudan granules presented an adjuvant efficacy on median motor nerve conduction velocity (SMD, 1.61; 95% CI, 1.16 to 2.07), median sensory nerve conduction velocity (SMD, 1.73; 95% CI, 1.26 to 2.20), common peroneal motor nerve conduction velocity (SMD, 1.48; 95% CI, 1.10 to 1.86), common peroneal sensory nerve conduction velocity (SMD, 1.57; 95% CI, 1.23 to 1.92), tibial motor nerve conduction velocity (SMD, 1.34; 95% CI, 0.82 to 1.87), and tibial sensory nerve conduction velocity (SMD, 1.03; 95% CI, 0.86 to 1.20). In terms of adverse events, there was no statistically significant difference between the trial group and the control group (P =0.87). Five preclinical studies were also retrieved for the study. Animal studies have shown that Mudan granules have anti-oxidative stress effects and could reduce the inflammatory response. It may improve peripheral nerve injury in diabetic rats by modulating the TLR4/MyD88/NF-{kappa}B pathway, TLR4/p38 MAPK pathway and PI3K/AKT pathway. ConclusionsMudan granules presented an adjuvant efficacy on patients with DPN and could improve the oxidative stress and inflammatory levels in preclinical models. However, high-quality original studies are needed to further prove the evidence.

Xeroderma pigmentosum (XP) is an inherited photoaging syndrome caused by mutations in genes involved in the nucleotide excision repair (NER) pathway. XP patients exhibit hypersensitivity to ultraviolet (UV) radiation, leading to accelerated skin aging and requiring lifelong sun avoidance. Here, we demonstrate that UV-induced DNA damage triggers cellular senescence and up-regulates senescence-associated secretory phenotype (SASP) genes in melanocytes derived from an XP patient. To explore the potential therapeutics for XP, we developed a cisplatin-based drug screening system and identified JAK inhibitors and curcuminoids as promising senomorphic agents. In addition, two classes of senolytic agents, BCL-2-like protein inhibitors and HSP90 inhibitors, effectively eliminate senescent melanocytes. Further analysis demonstrates that senomorphic treatment effectively counteracts senescence and reduces SASP gene expression in XP-derived melanocytes. Moreover, genes in senescence-related pathways, including the JAK/STAT, Type I interferon (IFN-I), and PI3K/AKT pathways, which are activated by both UV irradiation and cisplatin treatment, are down-regulated by senomorphic treatment. This study highlights a potential senotherapeutic strategy for XP, which may help alleviate photoaging symptoms in XP patients.

Down syndrome (DS) results from trisomy for human chromosome 21 and is the most frequent genetic cause of intellectual disability. No effective treatments currently exist that improve neurodevelopment and cognition. Atypical brain development in individuals with DS is apparent before birth, which suggests that the optimal time to begin administration of therapies is prenatally. Human neural progenitor cell (NPC) cultures provide a tractable in vitro model system to examine the effects of trisomy 21 (T21) on neurodevelopment and to measure the effects of pharmacological interventions. Here we report the results of preclinical studies evaluating 24 candidate therapies. RNA-Seq analyses found that euploid and T21 NPCs showed different transcriptomic responses to five candidate pharmacotherapies. The Rho-associated coiled-coil kinase (ROCK) inhibitor fasudil increased proliferation of T21 NPCs, reduced expression of inflammatory pathway genes in T21 NPCs, and reduced markers of inflammation in LPS-stimulated microglia model systems. These results demonstrate that fasudil can alter multiple T21-associated abnormalities in a beneficial manner, suggesting that fasudil warrants further study as a candidate prenatal pharmacotherapy for DS.

Introductory paragraphThe retinal pigmented epithelial (RPE) cells maintain retinal homeostasis, and alterations in their function contribute to non-exudative age-related macular degeneration (AMD)1,2. Here, we explore the intricate relationship between RPE cells, epigenetic modifications, and the development of AMD. Importantly, the study reveals a substantial decrease in histone deacetylase 3 (HDAC3) activity and elevated histone acetylation in the RPE of human AMD donor eyes. To investigate epigenetic mechanisms in AMD development, we used a mouse model with RPE-specific Cryba1 knockout3-5, revealing that the loss of {beta}A3/A1-crystallin selectively reduces HDAC3 activity, resulting in increased histone acetylation. {beta}A3/A1-crystallin activates HDAC3 by facilitating its interaction with the casein kinase II (CK2) and phosphorylating HDAC3, as well as by regulating intracellular InsP6 (phytic acid) levels, required for activating HDAC3. These findings highlight a novel function of {beta}A3/A1-crystallin as an epigenetic regulator of HDAC3 in the RPE cells and provide insights into potential therapeutic strategies in non-exudative AMD.