Cross-reaction mediated by distinct key amino acid combinations in the complementary-determining region (CDR) of a monoclonal antibody.

In immunology, cross-reaction between antigens and antibodies are commonly observed. Prior research has shown that various monoclonal antibodies (mAbs) can recognize a broad spectrum of epitopes related to influenza viruses. However, existing theories on cross-reactions fall short in explaining the phenomena observed. This study explored the interaction characteristics of H1-74 mAb with three peptides: two natural peptides, LVLWGIHHP and LPFQNI, derived from the hemagglutinin (HA) antigen of the H1N1 influenza virus, and one synthetic peptide, WPFQNY. Our findings indicate that the complementarity-determining region (CDR) of H1-74 mAb comprised five antigen-binding sites, containing eight key amino acid residues from the light chain variable region and 16 from the heavy chain variable region. These critical residues formed distinct hydrophobic or hydrophilic clusters and functional groups within the binding sites, facilitating interaction with antigen epitopes through hydrogen bonding, salt bridge formation, and π-π stacking. The study revealed that the formation of the antibody molecule led to the creation of binding groups and small units in the CDR, allowing the antibody to attach to a variety of antigen epitopes through diverse combinations of these small units and functional groups. This unique ability of the antibody to bind with antigen epitopes provides a new molecular basis for explaining the phenomenon of antibody cross-reaction.

Engineering and Purification of Microcin C7 Variants Resistant to Trypsin and Analysis of Their Biological Activity.

Microcin C7 (McC) as a viable form of antimicrobial has gained substantial attention due to its distinctive antimicrobial activity, by targeting aspartyl tRNA synthetase. McC can be a potential solution against pathogenic microbial infections in the postantibiotic era. However, considering that degradation by digestive enzymes can disrupt the function of this peptide in the gastrointestinal tract, in this study, we attempt to design McC variants to overcome several barriers that may affect its stability and biological activity. The mccA gene encoding the McC peptide precursor was mutated and 12 new McC variants with trypsin resistance were found. The Yej+rimL- strain was used as an indicator to determine the minimum inhibitory concentrations (MICs). The results showed that three variants, including R2A, R2T and R2Q, among 12 variants formed by the replacement of the second arginine of the McC peptide with different amino acids, were resistant to trypsin and had an outstanding antimicrobial ability, with MIC values of 12.5, 25, and 25 µg/mL, respectively. Taken together, our findings show that the engineering of the site-directed mutagenesis of McC significantly enhances McC trypsin resistance and maintains a great antimicrobial activity.

Multi-Omics Analysis Reveals the Gut Microbiota Characteristics of Diarrheal Piglets Treated with Gentamicin.

The involvement of alterations in gut microbiota composition due to the use of antibiotics has been widely observed. However, a clear picture of the influences of gentamicin, which is employed for the treatment of bacterial diarrhea in animal production, are largely unknown. Here, we addressed this problem using piglet models susceptible to enterotoxigenic Escherichia coli (ETEC) F4, which were treated with gentamicin. Gentamicin significantly alleviated diarrhea and intestinal injury. Through 16s RNS sequencing, it was found that gentamicin increased species richness but decreased community evenness. Additionally, clear clustering was observed between the gentamicin-treated group and the other groups. More importantly, with the establishment of a completely different microbial structure, a novel metabolite composition profile was formed. KEGG database annotation revealed that arachidonic acid metabolism and vancomycin resistance were the most significantly downregulated and upregulated pathways after gentamicin treatment, respectively. Meanwhile, we identified seven possible targets of gentamicin closely related to these two functional pathways through a comprehensive analysis. Taken together, these findings demonstrate that gentamicin therapy for diarrhea is associated with the downregulation of arachidonic acid metabolism. During this process, intestinal microbiota dysbiosis is induced, leading to increased levels of the vancomycin resistance pathway. An improved understanding of the roles of these processes will advance the conception and realization of new therapeutic and preventive strategies.

Immunomodulatory Effects of Microcin C7 in Cyclophosphamide-Induced Immunosuppressed Mice.

Microcin C7 (McC) as a viable immunomodulator peptide can be a potential solution for pathogenic microbial infection in the post-antibiotic era and has gained substantial attention. This study was designed to evaluate the immunomodulatory activity of Microcin C7 in a cyclophosphamide (CTX)-induced immunodeficient mouse model. We show that Microcin C7 treatment significantly alleviated the CTX-caused body weight loss, improved the feed and water consumption to improve the state of the mice, and elevated the absolute number and proportion of peripheral blood lymphocytes as well as the level of hemoglobulin. We further aim to characterize the phenotypes of the immune function and intestinal health profiles. The results demonstrate that Microcin C7 treatment increased serum levels of immunoglobulin A (IgA), IgG, interleukin 6, and hemolysin, promoted splenic lymphocyte proliferation induced by concanavalin A and LPS, and enhanced the phagocytosis of peritoneal macrophages immunized by sheep red blood cells. Additionally, Microcin C7 treatment decreased levels of diamine oxidase and d-lactate, ameliorated CTX-induced intestinal morphological damage, and increased the levels of zonula occluden 1, occludin, claudin-1, mucin 2, and secretary IgA in the jejunum and colon. Moreover, Microcin C7 administration is sufficient to reverse CTX-induced intestinal microbiota dysbiosis by increasing the number of Lactobacillus and Bifidobacterium, decreasing the number of Escherichia coli in colonic contents. Collectively, our results demonstrate that Microcin C7 may have protective and immunomodulatory functions and could be a potential candidate used in animal feed, functional foods, and immunological regimens..

pH/Temperature Responsive Curcumin-Loaded Micelle Nanoparticles Promote Functional Repair after Spinal Cord Injury in Rats via Modulation of Inflammation.

BACKGROUND: The formation of an inhibitory inflammatory microenvironment after spinal cord injury (SCI) remains a great challenge for nerve regeneration. The poor local microenvironment exacerbates nerve cell death; therefore, the reconstruction of a favorable microenvironment through small-molecule drugs is a promising strategy for promoting nerve regeneration. METHODS: In the present study, we synthesized curcumin-loaded micelle nanoparticles (Cur-NPs) to increase curcumin bioavailability and analyzed the physical and chemical properties of Cur-NPs by characterization experiments. We established an in vivo SCI model in rats and examined the ability of hind limb motor recovery using Basso-Beattie-Bresnahan scoring and hind limb trajectory assays. We also analyzed neural regeneration after SCI using immunofluorescence staining. RESULTS: The nanoparticles achieved the intelligent responsive release of curcumin while improving curcumin bioavailability. Most importantly, the released curcumin attenuated local inflammation by modulating the polarization of macrophages from an M1 pro-inflammatory phenotype to an M2 anti-inflammatory phenotype. M2-type macrophages can promote cell differentiation, proliferation, matrix secretion, and reorganization by secreting or expressing pro-repair cytokines to reduce the inflammatory response. The enhanced inflammatory microenvironment supported neuronal regeneration, nerve remyelination, and reduced scar formation. These effects facilitated functional repair in rats, mainly in the form of improved hindlimb movements. CONCLUSION: Here, we synthesized pH/temperature dual-sensitive Cur-NPs. While improving the bioavailability of the drug, they were also able to achieve a smart responsive release in the inflammatory microenvironment that develops after SCI. The Cur-NPs promoted the regeneration and functional recovery of nerves after SCI through anti-inflammatory effects, providing a promising strategy for the repair of SCIs.

3D collagen porous scaffold carrying PLGA-PTX/SDF-1α recruits and promotes neural stem cell differentiation for spinal cord injury repair.

Spinal Cord Injury (SCI), one of the major factors of disability, can cause irreversible motor and sensory impairment. There are no effective therapeutic drugs and technologies available in domestic or foreign countries currently. Neural stem cells (NSCs), with the potential for multidirectional differentiation, are a potential treatment for SCI. However, it has been demonstrated that NSCs primarily differentiated into astrocytes rather than neurons due to the inflammatory microenvironment, and the current challenge remains to direct the differentiation of NSCs into neurons in the lesion site. It was reported that the microtubule-stabilizing agent paclitaxel (PTX) was able to promote the differentiation of NSCs into neurons rather than astrocytes after SCI. SDF-1α can recruit NSCs and thus guide the migration of stem cells. In this study, we developed a functional collagen scaffold by loading SDF-1α and nanoparticle-encapsulated PLGA-PTX into a 3D collagen porous scaffold, allowing for slow release of PTX. When the functional scaffolds were implanted into the injury site, it provided a neural regeneration conduit channel for the migration of NSCs and neuronal differentiation. Neural regeneration promoted the recovery of motor function and reduced glial scar formation after SCI. In conclusion, a 3D collagen porous scaffold combined with PLGA-PTX and SDF-1α is a promising therapeutic strategy for SCI repair.

Disc regeneration by injectable fucoidan-methacrylated dextran hydrogels through mechanical transduction and macrophage immunomodulation.

Modulating a favorable inflammatory microenvironment that facilitates the recovery of degenerated discs is a key strategy in the treatment of intervertebral disc (IVD) degeneration (IDD). More interestingly, well-mechanized tissue-engineered scaffolds have been proven in recent years to be capable of sensing mechanical transduction to enhance the proliferation and activation of nucleus pulposus cells (NPC) and have demonstrated an increased potential in the treatment and recovery of degenerative discs. Additionally, existing surgical procedures may not be suitable for IDD treatment, warranting the requirement of new regenerative therapies for the restoration of disc structure and function. In this study, a light-sensitive injectable polysaccharide composite hydrogel with excellent mechanical properties was prepared using dextrose methacrylate (DexMA) and fucoidan with inflammation-modulating properties. Through numerous in vivo experiments, it was shown that the co-culture of this composite hydrogel with interleukin-1ß-stimulated NPCs was able to promote cell proliferation whilst preventing inflammation. Additionally, activation of the caveolin1-yes-associated protein (CAV1-YAP) mechanotransduction axis promoted extracellular matrix (ECM) metabolism and thus jointly promoted IVD regeneration. After injection into an IDD rat model, the composite hydrogel inhibited the local inflammatory response by inducing macrophage M2 polarization and gradually reducing the ECM degradation. In this study, we propose a fucoidan-DexMA composite hydrogel, which provides an attractive approach for IVD regeneration.

Dissecting Sperm Mitochondrial G-Quadruplex Structures Using a Fluorescent Probe Biomarker to Monitor and Regulate Fertilization Capability.

To monitor the levels of mitochondrial DNA G-quadruplexes (mtDNA G4s) in spermatozoa and to explore the possibility using mtDNA G4s as a reliable marker in patients with multiple clinical insemination failures, a novel chemical TPE-mTO probe engineered in our previous work was used on both samples from the mice sperm and from patients with fertilization failure. Expression of valosin-containing protein and the zona-free hamster egg assay were used to evaluate mitophagy and human sperm penetration. RNA-sequencing was used to explore expression changes of key genes affected by mtDNA G4s. Results showed that the probe can track mtDNA G4s in spermatozoa easily and quickly with fewer backgrounds. Significantly increased mtDNA G4s were also found in patients with fertilization failure, using the flow-cytometry-based TPE-mTO probe detection method. A sperm-hamster egg penetration experiment showed that abnormal fertilization caused by increased mtDNA G4s can be effectively restored by a mitophagy inducer. This study provides a novel method for monitoring etiological biomarkers in patients with clinical infertility and treatment for patients with abnormal fertilization caused by mtDNA G4 dysfunction.

Evaluation of Effectiveness and Safety of Microcin C7 in Weaned Piglets.

The effects and safety of dietary supplementation with Microcin C7 (C7) were evaluated in 216 weaned piglets. The pigs were given a control corn−soybean meal basal diet or C7 diet (control diet supplemented with 250, 500, 750, 1000, or 5000 mg C7/kg diets). Compared with the control group, the 500 mg/kg C7 supplementation group had better intestinal morphological indicators (p < 0.05), which may help maintain intestinal epithelial function and increase the growth performance (p < 0.05) and apparent total tract digestibility (p < 0.05). The diarrhea indexes of the 250, 500, and 750 mg/kg groups were significantly lower than that of the control group at 0−28 d (p < 0.05), and the 500 mg/kg group had the lowest diarrhea indexes (linear and quadratic, p < 0.05). A comprehensive analysis showed that microbial structure was significantly correlated with the degree of diarrhea, and the diarrhea-alleviating effect of C7 may be related to its selective regulation of specific microbial taxa. The 250 and 500 mg/kg C7 supplementation also significantly improved several immune indices of piglets (p < 0.05). Compared with the control diet, 5000 mg/kg C7 supplementation had no significant adverse effect on all parameters. Overall, the 250−500 mg/kg dose had the best effect, and the highest dose (5000 mg/kg) posed no toxicity risk. Therefore, C7 appears safe for use as an alternative to antibiotic growth promoters in weaned piglets.

Are we ready for the revision of the 14-day rule? Implications from Chinese legislations guiding human embryo and embryoid research.

The ISSCR recently released new guidelines that relaxed the 14-day rule taking away the tough barrier, and this has rekindled relevant ethical controversies and posed a fresh set of challenges to each nation's legislations and policies directly or indirectly. To understand its broad implications and the variation and impact of China's relevant national policies, we reviewed and evaluated Chinese laws, administrative regulations, departmental rules, and normative documents on fundamental and preclinical research involving human embryos from 1985 to 2022 in this paper. We have historically examined whether these regulations, including a 14-day rule, had restrictions on human embryo research, and whether and how these policies affected human embryo and embryoid research in China. We also discussed and assessed the backdrop in which China has endeavored to handle such as the need for expanding debates among justice practice, academia, and the public, and the shifting external environment influenced by fast-developing science and technology and people's culture and religions. In general, Chinese society commonly opposes giving embryos or fetuses the legal status of humans, presumably due to the Chinese public not seeming to have any strong religious beliefs regarding the embryo. On this basis, they do not strongly oppose the potential expansion of the 14-day rule. After the guidelines to strengthen governance over ethics in science, and technology were released by the Chinese government in 2022, Chinese policymakers have incorporated bioethics into the national strategic goals using a "People-Centered" approach to develop and promote an ecological civilization. Specifically, China follows the "precautionary principle" based on ethical priority as it believes that if scientific research carries any potential technological and moral risks on which no social ethical consensus has been attained, there would be a need to impose oversight for prevention and precaution. At the same time, China has adopted a hybrid legislative model of legislation and ethical regulations with criminal, civil and administrative sanctions and a 14-day limit specified within its national hESCs guidelines. This would certainly be a useful example for other countries to use when considering the possibility of developing a comprehensive, credible and sustainable regulatory framework.

The novel BRDT inhibitor NHWD870 shows potential as a male contraceptive in mice.

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) family proteins have emerged as promising options not only for the treatment of multiple cancers but also for disturbing the process of sperm maturation with potential for use as viable contraceptive targets. In this study, we find that the BET family inhibitor NHWD870 and BRDT can bind well in vitro through bioinformatics software prediction and protein binding inhibition experiments. NHWD870 can produce a good contraceptive effect through animal experiments in vivo, and the fertility can be restored to normal after drug withdrawal. Transcriptomics and proteomics results suggest that NHWD870 affects pathways related to spermatogenesis and maturation, further contributing to the male infertility phenotype. Our results show that NHWD870 can induce a complete and reversible contraceptive effect in mice, which is stronger than that of JQ1 and its synthesized derivatives. This study is expected to eventually lead to clinical trials.

Reconsidering the need for gain-of-function research on enhanced potential pandemic pathogens in the post-COVID-19 era.

The dual-use risk of infectious disease research using enhanced potential pandemic pathogens (ePPP), particularly gain-of-function (GOF) research, has been debated since 2011. As of now, research is supported on the condition that the research plan is reviewed and the actual experiment is supervised. However, the kinds of research conducted and what benefits they have brought to our society have not been adequately verified. Nevertheless, due to the COVID-19 pandemic that began at the end of 2019 and caused numerous deaths and wide economic disruption, the importance of infectious disease control from an international perspective has been recognized. Although complete control of the pandemic is still far off, positive signs include generating epidemiological trends based on genome analysis, therapeutic drug and vaccine development, clinical patient management, and public health policy interventions. In this context, the time has come to reconsider the true significance of GOF research on ePPP and the state of research governance in the post-COVID-19 era. In particular, the risks of such research are clearer than before, whereas its benefits seem less apparent. In this paper, we summarize the history of discussions on such GOF research, its significance in the light of the current COVID-19 pandemic, and the direction we shall take in the future.

Correlation between novel compound heterozygous ADAMTSL4 variants and primary phenotypes of ectopia lentis et pupillae.

PURPOSE: To investigate molecular pathogenesis of congenital ectopia lentis accompanied by various ophthalmic manifestations in a pedigree. METHODS: Three female siblings, their spouse and offspring underwent ophthalmic and general medical examinations. Genetic variants were screened with the whole exome sequencing and analyzed in either a dominant or recessive inheritance manner. Gene mutations were ascertained with the Sanger sequencing after the polymerase chain reaction. RESULTS: All three female siblings were diagnosed as the Ectopia lentis et pupillae (ELeP) through combination of clinical examination and genetic analysis. No characteristic pathological changes of skeletal, metabolic and cardiac abnormalities were observed. Thirteen genetic variants were selected out through analyzing in the dominant or recessive inheritance manner, but they were not associated with EL. Among them, ALOX15B variant may explain the skin disease in this pedigree. After inspection the known genes related to EL, novel compound heterozygous mutations (p.Ser264LeufsX37/p.Gly757ValfsX62) in ADAMTSL4 were discreetly identified in this ELeP pedigree. CONCLUSIONS: Novel compound heterozygous ADAMTSL4 variants are responsible for ELeP in the current pedigree. Correlation between ADAMTSL4 variants and ELeP was firstly established based on our 12 years follow-up studies and previous reports of ELeP and of ADAMTSL4-related eye disorders. The primary phenotypes caused by ADAMTSL4 variants include EL, EP, poor pupillary dilation, and axial elongation. Highly varying phenotypes including glaucoma, high myopia retinapathy, and poor vision and so on may be the secondary impairments. All these secondary impairments may be improved if proper clinical interventions are implemented in time.

Effectiveness and safety evaluation of graded levels of N-carbamylglutamate in growing-finishing pigs.

The aim of this study was as follows: 1) to investigate the effects of graded levels of N-carbamylglutamate (NCG) on performance, blood biochemical indexes, carcass traits and related indicators in growing-finishing pigs, and 2) to determine the optimal supplemental level. The toxicity of high-dose (much higher than recommended levels) NCG was assessed by routine blood tests and blood biochemical and histopathologic examinations of the heart, liver, spleen, lung, kidney and stomach. One hundred and forty-four growing-finishing pigs (Duroc × Large White × Landrace, 32.24 ± 1.03 kg) were used in a 74-d experiment and each treatment was replicated 6 times with 4 pigs (2 barrows and 2 gilts) per replicate. The dietary treatments were a corn-soybean meal basal diet supplemented with 0% (control), 0.05%, 0.1%, 0.15%, 0.2% or 1% NCG. The first 5 groups were used to explore the optimal supplemental level of NCG, while the control, 0.1% and 1% NCG groups were used to explore the safety of high-dose NCG. Compared with the normal control group, the final body weight and average daily gain tended to be higher in the 0.1% group (P = 0.08), the lean percentage tended to be higher in the 0.05% group (P = 0.07), the levels of free amino acids in the blood significantly increased in the 0.1% group (P < 0.05), both 0.1% and 0.15% NCG supplementation increased the levels of nitric oxide (NO) in serum (P = 0.07) and muscle growth- and lipid metabolism-related gene expression (P < 0.05) and NCG supplementation improved C18:1N9C monounsaturated fatty acids (MUFA) in a dose-dependent manner (P = 0.08). In addition, routine blood tests, blood biochemical indexes and histopathological examination revealed no abnormalities. Overall, increasing the levels of NCG did not linearly improve the above indicators; the 0.1% dose showed the best effect, and a high dose (1%) did not pose a toxicity risk.

Towards Better Governance on Biosafety and Biosecurity: China's Advances and Perspectives in Medical Biotechnology Legislation.

In this paper, we systematically investigated and assessed China's evolving medical biotechnology legislative and regulatory regime. 89 laws, rules, measures, guidelines, and views from 1985 to 2022 were systematically analogized and 28 were found to be involved in medical biotechnology legislation, including the recently passed Biosecurity Law. We classified the legislations and performed a comparative analysis for their legal binding based on the legal subject and extent of application, then further analyzes some of the legislative challenges in governing medical biotechnology risks in the context of China's upgrading its regulatory and legal regime in the last 3 years. We concluded that policymakers in China have now incorporated medical biotechnology-related biosafety and biosecurity into the national strategic goals of a "People-Centered" approach to establish and foster an ecological civilization, particularly in the aftermath of the "He Jiankui affair." Instead of relying on a patchwork of existing regulations and measures relating to the emerging field of medical biotechnology, China is attempting to integrate a patchwork of existing regulations and measures into a comprehensive legal framework, such as the constitution, National Security Law, Biosecurity Law, administrative regulations, departmental and local rules, and has begun to use the Civil Code and Criminal Law to explicitly identify actions relating to medical biotechnology. In general, China follows the "precautionary principle" as it thinks that uncertainty in science and technology should not be used to justify delaying the adoption of measures to prevent injuries or dangers, stating that whoever advances biotechnology must face the burden of proof of no harm. There would be a need to impose oversight for prevention and precaution if any biotechnology breakthrough that carries risks on which no scientific consensus has been reached. We argued that the "top-down" formulation of general objectives by the active political leadership and "bottom-up" innovation in the implementation are the keys to achieving these goals. Given the rapid advancements in medical biotechnology, countries all over the world must examine the governance landscape around biosafety and biosecurity and quickly consider options for their own comprehensive, credible, and long-lasting regulatory frameworks and experiences learned from China's governance will help chart a scalable future roadmap.

A loss-of-function variant in SSFA2 causes male infertility with globozoospermia and failed oocyte activation.

Globozoospermia (OMIM: 102530) is a rare type of teratozoospermia (< 0.1%). The etiology of globozoospermia is complicated and has not been fully revealed. Here, we report an infertile patient with globozoospermia. Variational analysis revealed a homozygous missense variant in the SSFA2 gene (NM_001130445.3: c.3671G > A; p.R1224Q) in the patient. This variant significantly reduced the protein expression of SSFA2. Immunofluorescence staining showed positive SSFA2 expression in the acrosome of human sperm. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and Coimmunoprecipitation (Co-IP) analyses identified that GSTM3 and Actin interact with SSFA2. Further investigation revealed that for the patient, regular intracytoplasmic sperm injection (ICSI) treatment had a poor prognosis. However, Artificial oocyte activation (AOA) by a calcium ionophore (A23187) after ICSI successfully rescued the oocyte activation failure for the patient with the SSFA2 variant, and the couple achieved a live birth. This study revealed that SSFA2 plays an important role in acrosome formation, and the homozygous c.3671G > A loss-of-function variant in SSFA2 caused globozoospermia. SSFA2 may represent a new gene in the genetic diagnosis of globozoospermia, especially the successful outcome of AOA-ICSI treatment for couples, which has potential value for clinicians in their treatment regimen selections.

Governance of Heritable Human Gene Editing World-Wide and Beyond.

To date, the controversy surrounding the unknown risks and consequences of heritable genome editing has grown, with such work raising biosafety and ethical concerns for future generations. However, the current guideline of global governance is limited. In the context of the new framework for the governance of human genome editing developed by the World Health Organization (WHO) committee, this paper presents further analysis by highlighting predicaments of governance on germline engineering that merit the most attention: (1) building a scientific culture informed by a broader set of values and considerations in the internal scientific community at large, such as codes of ethics, and education, in addition to awareness-raising measures; and (2) reflecting on and institutionalizing policies in grassroots practice according to local conditions in external governance, such as the experimentalist governance, which is a multi-layered model of governance that establishes an open-ended framework from the top and offers stakeholders the freedom of discussion. The key to achieving these goals is more democratic deliberation between the public and the inclusive engagement of the global scientific community, which has been extensively used in the Biological and Toxin Weapons Convention (BTWC). On a global scale, we believe that practicing heritable human genome editing in accordance with the WHO and BTWC appears to be a good choice.