Discovery and evolution of berberine analogues as anti-Helicobacter pylori agents with multi-target mechanisms.

Thirty protoberberine derivatives, of which twenty five were new, were synthesized and evaluated for their anti-Helicobacter pylori (HP) activities, taking 2,3,10-trimethoxy-9-p-methylbenzylaminoprotopalmatine chloride 1 as the lead. Among them, berberine (BBR) derivative 7c displayed the highest potency against six tested metronidazole (MTZ)-resistant strains and two tested MTZ-susceptible strains with the MIC values of 0.4-1.6 µg/mL with favorable druglike profiles including low toxicity and high stabilities in plasma and artificial gastric fluid. Mechanistic study revealed that 7c might target HP urease with IC50 value of 0.27 µg/mL against Jack bean urease. Furthermore, 7c might change the permeability of the bacterial membrane and direct interact with HP DNA, which also contribute to its bactericidal activity. Therefore, BBR derivatives constituted a new family of anti-HP candidates, with the advantage of good safety profile and multi-target mechanisms, and are worthy for further investigation.

AC81 Is a Putative Disulfide Isomerase Involved in Baculoviral Disulfide Bond Formation.

The correct formation of native disulfide bonds is critical for the proper structure and function of many proteins. Cellular disulfide bond formation pathways commonly consist of two parts: sulfhydryl oxidase-mediated oxidation and disulfide isomerase-mediated isomerization. Some large DNA viruses, such as baculoviruses, encode sulfhydryl oxidases, but viral disulfide isomerases have not yet been identified, although G4L in poxvirus has been suggested to serve such a function. Here, we report that the baculovirus core gene ac81 encodes a putative disulfide isomerase. ac81 is conserved in baculoviruses, nudiviruses, and hytrosaviruses. We found that AC81 homologs contain a typical thioredoxin fold conserved in disulfide isomerases. To determine the role of AC81, a series of Autographa californica nucleopolyhedrovirus (AcMNPV) bacmids containing ac81 knockout or point mutations was generated, and the results showed that AC81 is essential for budded virus production, multinucleocapsid occlusion-derived virus (ODV) formation, and ODV embedding in occlusion bodies. Nonreducing Western blot analysis indicated that disulfide bond formation in per os infectivity factor 5 (PIF5), a substrate of the baculoviral sulfhydryl oxidase P33, was abnormal when ac81 was knocked out or mutated. Pulldown assays showed that AC81 interacted with PIF5 and P33 in infected cells. In addition, two critical regions that harbor key amino acids for function were identified in AC81. Taken together, our results suggest that AC81 is a key component involved in the baculovirus disulfide bond formation pathway and likely functions as a disulfide isomerase. IMPORTANCE Many large DNA viruses, such as poxvirus, asfarvirus, and baculovirus, encode their own sulfhydryl oxidase to facilitate the disulfide bond formation of viral proteins. Here, we show that AC81 functions as a putative disulfide isomerase and is involved in multiple functions of the baculovirus life cycle. Interestingly, AC81 and P33 (sulfhydryl oxidase) are conserved in baculoviruses, nudiviruses, and hytrosaviruses, which are all insect-specific large DNA viruses replicating in the nucleus, suggesting that viral disulfide bond formation is an ancient mechanism shared by these viruses.

Insights into Two-Metal-Ion Catalytic Mechanism of Cap-Snatching Endonuclease of Ebinur Lake Virus in Bunyavirales.

The cap-snatching endonuclease (EN) of segmented negative-strand RNA viruses (sNSVs) produces short capped primers for viral transcription by cleaving the host mRNAs. EN requires divalent metals as cofactors for nucleic acid substrates cleavage; however, the detailed mechanism of metal ion-dependent catalysis of ENs remains obscure. In this work, we reported the EN crystal structure of the Ebinur Lake virus (EBIV), an emerging mosquito-borne orthobunyavirus, and investigated its enzymatic properties and metal ion-based catalytic mechanism. In vitro biochemical data showed that EBIV EN is a specific RNA nuclease and prefers to cleave unstructured uridine-rich ssRNA. Structural comparison indicated that the overall structural architecture of EBIV EN is similar to that of other sNSV ENs, while the detailed active site configuration including the binding state of metal ions and the conformation of the LA/LB loop pair is different. Based on sequence conservation analysis, nine active site mutants were constructed, and seven crystal structures of them were determined. Mutations of active site residues associated with the two metal ions (Mn1 and Mn2) coordination abolished EN activity. Crystallographic analyses further revealed that none of these mutants bound two metal ions simultaneously in the active site. Importantly, we found that the perturbation of Mn1-coordination (metal site 1), resulted in the enhancement or elimination of Mn2-coordination (metal site 2). Taken together, our data provide structural evidence to support the two-metal-ion catalytic mechanism of EBIV EN and the correlation of metal binding at the two binding sites, which may be commonly shared by bunyaviruses or other sNSVs. IMPORTANCE The viral endonucleases (ENs) encoded by bunyaviruses and orthomyxoviruses play an essential role in initiating transcription by "snatching" capped primers from the host mRNAs. These ENs are metal-ion-dependent nucleases; however, the details of their catalytic mechanism remain elusive. Here, we reported high-resolution crystal structures of the wild-type and mutant ENs of a novel bunyavirus, the Ebinur Lake virus (EBIV), and revealed the structure and function relationship of EN. The EBIV EN exhibited differences in the details of active site structure compared to its homologues. Our data provided structural evidence to support a two-metal-ion catalytic mechanism of EBIV EN, and found the correlation of metal binding at both binding sites, which might reflect the dynamic structural properties that correlate to EN catalytic function. Taken together, our results revealed the structural characteristics of EBIV EN and made important implications for understanding the catalytic mechanism of cap-snatching ENs.

Structural and Biochemical Basis for Development of Diketo Acid Inhibitors Targeting the Cap-Snatching Endonuclease of the Ebinur Lake Virus (Order: Bunyavirales).

The Bunyavirales contain many important human pathogens that lack an antiviral therapy. The cap-snatching endonuclease (EN) of segmented negative-strand RNA viruses is an attractive target for broad-spectrum antivirals due to its essential role in initiating viral transcription. L-742,001, a previously reported diketo acid inhibitor against influenza virus EN, demonstrated potent EN inhibition and antiviral activity on various bunyaviruses. However, the precise inhibitory mechanism of the compound is still poorly understood. We recently characterized a highly active EN from Ebinur Lake virus (EBIV), a newly identified member of the Orthobunyavirus genus, and obtained its high-resolution structures, paving the way for structure-guided inhibitor development. Here, nine L-742,001 derivatives were designed and synthesized de novo, and their structure-activity relationship with EBIV EN was studied. In vitro biochemical data showed that the compounds inhibited the EBIV EN activity with different levels and could be divided into three categories. Five representative compounds were selected for further cell-based antiviral assay, and the results largely agreed with those of the EN assays. Furthermore, the precise binding modes of L-742,001 and its derivatives in EN were revealed by determining the high-resolution crystal structures of EN-inhibitor complexes, which suggested that the p-chlorobenzene is essential for the inhibitory activity and the flexible phenyl has the greatest exploration potential. This study provides an important basis for the structure-based design and optimization of inhibitors targeting EN of segmented negative-strand RNA viruses. IMPORTANCE The Bunyavirales contain many important human pathogens such as Crimean-Congo hemorrhagic fever virus and Lassa virus that pose serious threats to public health; however, currently there are no specific antiviral drugs against these viruses. The diketo acid inhibitor L-742,001 is a potential drug as it inactivates the cap-snatching endonuclease (EN) encoded by bunyaviruses. Here, we designed and synthesized nine L-742,001 derivatives and assessed the structure-activity relationship using EN of the newly identified Ebinur Lake virus (EBIV) as a research model. Our results revealed that the p-chlorobenzene of this broad-spectrum EN inhibitor is crucial for the inhibitory activity and the flexible phenyl "arm" has the best potential for further optimization. As cap-snatching ENs are present not only in bunyaviruses but also in influenza viruses, our data provide important guidelines for the development of novel and more potent diketo acid-based antiviral drugs against those viruses.

Per Os Infectivity Factor 5 Identified as a Substrate of P33 in the Baculoviral Disulfide Bond Formation Pathway.

Disulfide bonds are critical for the structure and function of many proteins. Some large DNA viruses encode their own sulfhydryl oxidase for disulfide bond formation. Previous studies have demonstrated that the baculovirus-encoded sulfhydryl oxidase P33 is necessary for progeny virus production, and its enzymatic activity is important for morphogenesis and oral infectivity of baculoviruses. However, the downstream substrates of P33 in the putative redox pathway of baculoviruses are unknown. In this study, we showed that PIF5, one of the per os infectivity factors (PIFs), contained intramolecular disulfide bonds and that the disulfide bond formation was interrupted in the absence of P33. In vivo pulldown and colocalization analyses revealed that PIF5 and P33 interacted with each other during virus infection. Further, in vitro assays validated that the reduced PIF5 proteins could be oxidized by P33. To understand the contribution of disulfide bonds to the function of PIF5, several cysteine-to-serine mutants were constructed, which all interfered with the disulfide bond formation of PIF5 to different extents. All the mutants lost their oral infectivity but had no impact on infectious budding virus (BV) production or virus morphogenesis. Taken together, our results indicated PIF5 as the first identified substrate of P33. Further, the disulfide bonds in PIF5 play an essential role in its function in oral infection.IMPORTANCE Similar to some large DNA viruses that encode their own disulfide bond pathway, baculovirus encodes a viral sulfhydryl oxidase, P33. Enzyme activity of P33 is related to infectious BV production, occlusion-derived virus (ODV) envelopment, occlusion body morphogenesis, and oral infectivity, suggesting that P33 is involved in disulfide bond formation of multiple proteins. A complete disulfide bond formation pathway normally contains a sulfhydryl oxidase, a disulfide-donating enzyme, and one or more substrates. In baculovirus, apart from P33, other components of the putative pathway remain unknown. In this study, we identified PIF5 as the first substrate of P33, which is fundamental for revealing the complete disulfide bond formation pathway in baculovirus. PIF5 is essential for oral infection and is absent from the PIF complex. Our study demonstrated that native disulfide bonds in PIF5 are required for oral infection, which will help us to reveal its mode of action.

Cholesterol biosynthesis supports the growth of hepatocarcinoma lesions depleted of fatty acid synthase in mice and humans.

OBJECTIVE: Increased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC). DESIGN: We investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies. RESULTS: Ablation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture. CONCLUSION: Our study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function.

Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity.IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

Zoledronate dysregulates fatty acid metabolism in renal tubular epithelial cells to induce nephrotoxicity.

Zoledronate is a bisphosphonate that is widely used in the treatment of metabolic bone diseases. However, zoledronate induces significant nephrotoxicity associated with acute tubular necrosis and renal fibrosis when administered intravenously. There is speculation that zoledronate-induced nephrotoxicity may result from its pharmacological activity as an inhibitor of the mevalonate pathway but the molecular mechanisms are not fully understood. In this report, human proximal tubular HK-2 cells and mouse models were combined to dissect the molecular pathways underlying nephropathy caused by zoledronate treatments. Metabolomic and proteomic assays revealed that multiple cellular processes were significantly disrupted, including the TGFß pathway, fatty acid metabolism and small GTPase signaling in zoledronate-treated HK-2 cells (50 µM) as compared with those in controls. Zoledronate treatments in cells (50 µM) and mice (3 mg/kg) increased TGFß/Smad3 pathway activation to induce fibrosis and kidney injury, and specifically elevated lipid accumulation and expression of fibrotic proteins. Conversely, fatty acid transport protein Slc27a2 deficiency or co-administration of PPARA agonist fenofibrate (20 mg/kg) prevented zoledronate-induced lipid accumulation and kidney fibrosis in mice, indicating that over-expression of fatty acid transporter SLC27A2 and defective fatty acid ß-oxidation following zoledronate treatments were significant factors contributing to its nephrotoxicity. These pharmacological and genetic studies provide an important mechanistic insight into zoledronate-associated kidney toxicity that will aid in development of therapeutic prevention and treatment options for this nephropathy.

Structure-function analysis of the ATPase domain of African swine fever virus topoisomerase.

Type II topoisomerase utilizes the energy from ATP hydrolysis to alter DNA topology during genome replication and transcription. The ATPase domain of this enzyme is required for ATP hydrolysis and plays a crucial role in coupling DNA binding and ATP turnover with the DNA strand passage reaction. The African swine fever virus (ASFV) specifically encodes a topoisomerase II (topo II), which is critical for viral replication and an attractive target for antiviral development. Here, we present a high-resolution crystal structure of the ASFV topo II ATPase domain complexed with the substrate analog AMPPNP. Structural comparison reveals that the ASFV topo II ATPase domain shares a conserved overall structure with its homologs from eukaryotes and prokaryotes but also has three characteristic regions, including the intra-molecular interface formed by the ATP-lid and QTK loop as well as helix α9, the K-loop in the transducer domain, and the antennae-like α-helix at the ATP binding domain. Mutating the key residues within these three regions impairs or abolishes the basal and DNA-stimulated ATPase activities and reduces or eliminates the relaxation activity of the holoenzyme. Our data indicate that all three regions are functionally important for the ATPase and relaxation activities and strongly suggest that ATP hydrolysis, DNA binding, and strand passage are highly coupled and managed by the allosteric coordination of multiple domains of the type II topoisomerase. Moreover, we find a promising druggable pocket in the dimeric interface of the ASFV topo II ATPase domain, which will benefit future anti-ASFV drug development. IMPORTANCE: The ATPase domain of type II topoisomerase provides energy by hydrolyzing ATP and coordinates with the DNA-binding/cleavage domain to drive and control DNA transport. The precise molecular mechanisms of how these domains respond to DNA binding and ATP hydrolysis signals and communicate with each other remain elusive. We determine the first high-resolution crystal structure of the ATPase domain of African swine fever virus (ASFV) topo II in complex with AMPPNP and biochemically investigate its function in ATPase and DNA relaxation activities. Importantly, we find that mutations at three characteristic regions of the ASFV ATPase domain produce parallel effects on the basal/DNA-stimulated ATPase and relaxation activities, implying the tight coupling of the ATP hydrolysis and strand passage process. Therefore, our data provide important implications for understanding the strand passage mechanism of the type II topoisomerase and the structural basis for developing ATPase domain-targeting antivirals against ASFV.

Polystyrene microplastics induce anxiety via HRAS derived PERK-NF-κB pathway.

Exposure to environmentally hazardous substances is recognized as a significant risk factor for neurological associated disorders. Among these substances, polystyrene microplastics (PS-MPs), widely utilized in various consumer products, have been reported to exhibit neurotoxicity. However, the potential association of PS-MPs with abnormal anxiety behaviors, along with the underlying molecular mechanisms and key proteins involved, remains insufficiently explored. Here, we delineated the potential mechanisms of PS-MPs-induced anxiety through proteomics and molecular investigations. We characterized the PS-MPs, observed their accumulation in the brain, leading to anxiety-like behavior in mice, which is correlated with microglia activation and pro-inflammatory response. Consistent with these findings, our studies on BV2 microglia cells showed that PS-MPs activated NF-κB-mediated inflammation resulting in the upregulation of pro-inflammatory cytokines such as TNFα and IL-1ß. Of particular significance, HRAS was identified as a key factor in the PS-MPs induced pro-inflammatory response through whole proteomics analysis, and knockdown of H-ras effectively inhibited PS-MPs induced PERK-NF-κB activation and associated pro-inflammatory response in microglia cells. Collectively, our findings highlight that PS-MPs induce anxiety of mice via the activation of the HRAS-derived PERK-NF-κB pathway in microlglia. Our results contribute valuable insights into the molecular mechanisms of PS-MPs-induced anxiety, and may offer implications for addressing neurotoxicity and prevention the adverse effects of environmentally hazardous substances, including microplastics.

Identification of miR-221 and -222 as important regulators in genotype IV swine hepatitis E virus ORF3-expressing HEK 293 cells.

Hepatitis E virus (HEV) has emerged as an important cause of epidemic and sporadic acute viral hepatitis worldwide, which is a major public health challenge. A better understanding of the interaction between the virus and the host cell would be very helpful for its therapy. Swine HEV (SHEV) open reading frame 3 (ORF3) is a regulatory protein that alters the activity of selected transcription factors and cytoplasmic signaling pathways. MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein-coding genes and represent an interesting lead to study SHEV infection and to identify new therapeutic targets. To explore how SHEV ORF3 affects miRNAs in host cells, we used miRNA array analysis to compare the expression patterns of miRNAs in stable cell lines that expressed or did not express SHEV ORF3. We found a significant down-regulation of miR-221 and -222 in ORF3 expressing human embryonic kidney 293 cell line. Among the 116 candidate targets genes of miR-221 and -222 that we detected in silico, we demonstrated that the expression of the cyclin-dependent kinase inhibitor 1B, also named p27(kip1), was directly regulated by these miRNAs. We hypothesize that SHEV ORF3-induced miR-221/222 downregulation enhances p27(kip1) expression in HEK293 cells. This provides new avenues for future exploration of the precise roles of miRNAs in SHEV infection.

Cryo-EM structures of African swine fever virus topoisomerase.

IMPORTANCE: African swine fever virus (ASFV) is a highly contagious virus that causes lethal hemorrhagic diseases known as African swine fever (ASF) with a case fatality rate of 100%. There is an urgent need to develop anti-ASFV drugs. We determine the first high-resolution structures of viral topoisomerase ASFV P1192R in both the closed and open C-gate forms. P1192R shows a similar overall architecture with eukaryotic and prokaryotic type II topoisomerases, which have been successful targets of many antimicrobials and anticancer drugs, with the most similarity to yeast topo II. P1192R also exhibits differences in the details of active site configuration, which are important to enzyme activity. These two structures offer useful structural information for antiviral drug design and provide structural evidence to support that eukaryotic type IIA topoisomerase likely originated from horizontal gene transfer from the virus.

Celastrol induces premature ovarian insufficiency by inducing apoptosis in granulosa cells.

Celastrol, a natural compound purified from the Chinese herb Tripterygium wilfordii Hook. f., has excellent pharmacological activity for the treatment of various diseases. Assessing the safety of its use is essential for its development into a clinical medicine. However, research assessing its toxicity on the female reproductive system has never been reported. In this study, the ovarian toxicity of celastrol and its underlying mechanism were investigated. We found that celastrol induced premature ovarian insufficiency and apoptosis in granulosa cells. Activity-based protein profiling results showed that high mobility group box 1 was a candidate target protein of celastrol. Celastrol directly bound to Cys106 of high mobility group box 1. Knocking down high mobility group box 1 induced apoptosis of granulosa cells, while overexpression of this gene reversed celastrol-induced apoptosis. Celastrol treatment upregulated p21 transcription, but overexpression of high mobility group box 1 reversed this upregulation. Thus, Celastrol induces premature ovarian insufficiency and apoptosis in granulosa cells by directly binding to high mobility group box 1 and interfering with its biological function to regulate p21 transcription. This study provides valuable information for assessing the safety of the clinical application of celastrol on female patients.

Dual roles and evolutionary implications of P26/poxin in antagonizing intracellular cGAS-STING and extracellular melanization immunity.

P26, a homolog of the viral-encoded nuclease poxin that neutralizes the cGAS-STING innate immunity, is widely distributed in various invertebrate viruses, lepidopteran insects, and parasitoid wasps. P26/poxin from certain insect viruses also retains protease activity, though its biological role remains unknown. Given that many P26s contain a signal peptide, it is surmised that P26 may possess certain extracellular functions. Here, we report that a secretory baculoviral P26 suppresses melanization, a prominent insect innate immunity against pathogen invasion. P26 targets the cofactor of a prophenoloxidase-activating protease, and its inhibitory function is independent of nuclease activity. The analysis of P26/poxin homologs from different origins suggests that the ability to inhibit the extracellular melanization pathway is limited to P26s with a signal peptide and not shared by the homologs without it. These findings highlight the independent evolution of a single viral suppressor to perform dual roles in modulating immunity during virus-host adaptation.

Crystal structure of the mismatch-specific thymine glycosylase domain of human methyl-CpG-binding protein MBD4.

Methyl-CpG (mCpG) binding domain protein 4 (MBD4) is a member of mammalian DNA glycosylase superfamily. It contains an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain, which is an important molecule believed to be involved in maintaining of genome stability. Herein, we determined the crystal structure of C-terminal glycosylase domain of human MBD4. And the structural alignments of other helix-hairpin-helix (HhH) DNA glycosylases show that the human MBD4 glycosylase domain has the similar active site and the catalytic mechanisms as others. But the different residues in the N-terminal of domain result in the change of charge distribution on the surface of the protein, which suggest the different roles that may relate some diseases.

Unexpected death in a young man associated with a unilateral swollen leg: Pathological and toxicological findings in a fatal snakebite from Deinagkistrodon acutus (Chinese moccasin).

Deinagkistrodon acutus (D. acutus), also known as the Chinese moccasin, is a viper species found throughout the southeastern parts of China, northern Vietnam and Laos. D. acutus envenomation can result in coagulopathy and lead to death if not treated correctly. A 20-year-old man was discovered with a severely swollen left thigh with overlying dark purple, discolored skin. He was immediately transported to hospital. Laboratory examinations revealed dysfunctional coagulation and fluid-electrolyte imbalances. He died 2 h later despite resuscitation efforts. Surveillance footage revealed that he had walked through a grass field while returning home that night. Autopsy and pathological examination findings revealed a large area of muscle necrosis of the left thigh, renal tubular necrosis, and hepatocyte necrosis. Potential fang marks were found on the decedent's jeans. Due to our suspicions, we performed specific enzyme-linked immunosorbent assays (ELISA) and detected D. acutus venom in the kidneys, left thigh muscle, liver, lung, spleen, and heart tissues of the decedent. In conclusion, the clinical manifestations, autopsy, histopathological examination, ELISA, and investigation results confirmed D. acutus envenomation.

Efficient endothelial and smooth muscle cell differentiation from human pluripotent stem cells through a simplified insulin-free culture system.

A major obstacle for using human pluripotent stem cells (hPSCs) derived vascular cells for cell therapy is the lack of simple, cost-saving, and scalable methods for cell production. Here we described a simplified and chemically defined medium (AATS) for endothelial cells (ECs) and smooth muscle cells (SMCs) differentiation. AATS medium does not contain insulin, enabling the rapid and highly efficient vascular mesoderm formation through accelerating metabolic and autophagy-enhanced mesoderm induction. Transcriptome profiling confirmed that hPSC-derived ECs and SMCs in the AATS medium closely resembled primary ECs and SMCs formed in vivo. ECs appeared to adhere and grow better in the AATS medium over other cell types, which allowed the purification of CD31+CD144+ double-positive cells. Furthermore, the AATS medium was compatible with 3D microscaffold (MS) culture, which may facilitate large-scale bioproduction of ECs. HPSC-derived ECs and SMCs in the AATS medium exhibited strong revascularization potential in treating murine ischemic models. Our study provided a cost-effective and efficient medium system to manufacture GMP compatible, off-the-shelf ECs, and SMCs to model human diseases and vascular repair.

SLC22A14 is a mitochondrial riboflavin transporter required for sperm oxidative phosphorylation and male fertility.

Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by genetic, biochemical, multi-omic, and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we find that fatty acid ß-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for the development of male contraceptives.

Synthesis, characterization, DNA binding, topoisomerase I inhibition and antiproliferation activities of three new functionalized terpyridine platinum(II) complexes.

Three new platinum(II) complexes with pendent morpholine were synthesized, namely complex 1 ([Pt(L)Cl]CF3SO3), complex 2 ([Pt(L)(NH3)](CF3SO3)2) and complex 3 ([Pt(L)(PPh3)](CF3SO3)2), where L = 4'-[4-(4-morpholinobutyloxy)phenyl]-2,2':6',2″-terpyridine and PPh3 = triphenylphosphine. The detailed molecular structures of complex 3, L and its precursor L' (1,4'-[4-(4-bromobutyloxy)phenyl]-2,2':6',2″-terpyridine) were determined by single crystal X-ray diffraction. An evaluation of in vitro cytotoxicity for both ligand and complexes was performed by methyl thiazolyl tetrazolium (MTT) assay in three cancer cell lines and normal cells as the control, respectively. IC50 values of complexes 1-3 were lower than those exhibited for the reference drug cisplatin, and selectivity of these complexes were greater than cisplatin. Among them, complex 3 with a leaving group PPh3 was found to be the most efficacious complex against certain cell lines, especially for cisplatin-resistant A549cisR cells. These complexes were found to bind DNA, induce efficient DNA unwinding. Meanwhile, topoisomerase (Topo) I inhibitory activities by three complexes were detected, and a minimum inhibitory concentration of 15 µM of complex 3 was found totally inhibit Topo I activity.

The cysteine-rich region of a baculovirus VP91 protein contributes to the morphogenesis of occlusion bodies.

The baculovirus core gene vp91 has been reported to be essential for nucleocapsid assembly and oral infection. Here, we studied the function of vp91 by analyzing its homologue, ha76, in Helicoverpa armigera nucleopolyhedrovirus (HearNPV). HA76 was expressed at the late stage of HearNPV infection; deletion of ha76 showed that the gene is required for budded virus production. A series of recombinants with truncated ha76 was constructed and analyzed in vitro and in vivo. The results showed that the region encoding the C-terminus of HA76 was essential for nucleocapsid assembly, whereas the N-terminal cysteine-rich region was responsible for oral infection. Electron microscope analyses further showed that the cysteine-rich region contributed to morphogenesis of occlusion bodies (OBs), with amino acids 136-223 of HA76 being critical for this function. The results revealed a novel function of VP91 and suggested that the impact on OB morphogenesis is partially related to oral infectivity.