A case of a lidocaine intoxication of a 12 week old boy due to a circumcision in a domestic setting - clinical, toxicological and forensic aspects.

Lidocaine is a local anaesthetic commonly used during circumcision for dorsal penile nerve block (DPNB). We describe a case of a 12-week-old infant who presented generalized seizures due to local anesthetic systemic toxicity after Lidocaine administration for circumcision in a non-hospital setting. Serum concentrations of Lidocaine (16.4 mg/L) and its main active metabolite monoethylglycinexylidide (MEGX, 1.36 mg/L) were determined by HPLC-DAD, in a sample collected shortly after administration, which were higher than in comparable cases reported in literature. The reason for the overdose was assumed to be accidental systemic application. Due to suspicion of an improperly performed circumcision and bodily harm, police was involved and a clinical forensic examination was carried out. Here, we present analytical, clinical and forensic aspects of this case.

Differences in the pathogenicity and molecular characteristics of fowl adenovirus serotype 4 epidemic strains in Guangxi Province, southern China.

Starting in 2015, the widespread prevalence of hydropericardium-hepatitis syndrome (HHS) has led to considerable financial losses within China's poultry farming industry. In this study, pathogenicity assessments, whole-genome sequencing, and analyses were conducted on 10 new isolates of the novel genotype FAdV-4 during a HHS outbreak in Guangxi Province, China, from 2019 to 2020. The results indicated that strains GX2019-010 to GX2019-013 and GX2019-015 to GX2019-018 were highly virulent, while strain GX2020-019 exhibited moderate virulence. Strain GX2019-014 was characterized as a wild-type strain with low virulence, displaying no pathogenic effects when 0.5 mL containing 106 TCID50 virus was inoculated into the muscle of specific pathogen-free (SPF) chickens at 4 weeks of age, while 107 TCID50 and 108 TCID50 resulted in mortality rates of 80 and 100%, respectively. The whole genomes of strains GX2019-010 to GX2019-013, GX2019-015 to GX2019-018, and GX2020-019 showed high homology with other Chinese newly emerging highly pathogenic FAdV-4 strains, whereas GX2019-014 was closer to nonmutant strains and shared the same residues with known nonpathogenic strains (B1-7, KR5, and ON1) at positions 219AA and 380AA of the Fiber-2 protein. Our work enriches the research on prevalent strains of FAdV-4 in China, expands the knowledge on the virulence diversity of the novel genotype FAdV-4, and provides valuable reference material for further investigations into the key virulence-associated genetic loci of FAdV-4.

Influence of Gadoxetate disodium to the hepatic proton density fat fraction quantified with the Dixon sequences in a rabbit model.

OBJECTIVE: To study the impact of Gx on quantification of hepatic fat contents under metabolic dysfunction-associated steatotic liver disease (MASLD) imaged on VIBE Dixon in hepatobiliary specific phase. METHODS: Forty-two rabbits were randomly divided into control group (n = 10) and high-fat diet group (n = 32). Imaging was performed before enhancement (Pre-Gx) and at the 13th (Post-Gx13) and 17th (Post-Gx17) min after Gx enhancement with 2E- and 6E-VIBE Dixon to determine hepatic proton density fat fractions (PDFF). PDFFs were compared with vacuole percentage (VP) measured under histopathology. RESULTS: 33 animals were evaluated and including control group (n = 11) and MASLD group (n = 22). Pre-Gx, Post-Gx13, Post-Gx17 PDFFs under 6E-VIBE Dixon had strong correlations with VPs (r2 = 0.8208-0.8536). PDFFs under 2E-VIBE Dixon were reduced significantly (P < 0.001) after enhancement (r2 = 0.7991/0.8014) compared with that before enhancement (r2 = 0.7643). There was no significant difference between PDFFs of Post-Gx13 and Post-Gx17 (P = 0.123) for which the highest consistency being found with 6E-VIBE Dixon before enhancement (r2 = 0.8536). The signal intensity of the precontrast compared with the postcontrast, water image under 2E-VIBE Dixon increased significantly (P < 0.001), fat image showed no significant difference (P = 0.754). CONCLUSION: 2E- and 6E-VIBE Dixon can obtain accurate PDFFs in the hepatobiliary specific phase from 13 to 17th min after Gx enhancement. On 2E-VIBE Dixon (FA = 10°), effective minimization of T1 Bias by the Gx administration markedly improved the accuracy of the hepatic PDFF quantification.

Splenic proteome profiling in response to Marek's disease virus strain GX0101 infection.

Marek's disease virus (MDV) strain GX0101 was the first reported field strain of recombinant gallid herpesvirus type 2 (GaHV-2). However, the splenic proteome of MDV-infected chickens remains unclear. In this study, a total of 28 1-day-old SPF chickens were intraperitoneally injected with chicken embryo fibroblast (CEF) containing 2000 PFU GX0101. Additionally, a control group, consisting of four one-day-old SPF chickens, received intraperitoneal equal doses of CEF. Blood and various tissue samples were collected at different intervals (7, 14, 21, 30, 45, 60, and 90 days post-infection; dpi) for histopathological, real-time PCR, and label-free quantitative analyses. The results showed that the serum expressions of MDV-related genes, meq and gB, peaked at 45 dpi. The heart, liver, and spleen were dissected at 30 and 45 dpi, and their hematoxylin-eosin staining indicated that virus infection compromised the normal organizational structure at 45 dpi. Particularly, the spleen structure was severely damaged, and the lymphocytes in the white medulla were significantly reduced. Furthermore, liquid chromatography-mass spectrometry (LC-MS) and label-free techniques were used to analyze the difference in splenic proteome profiles of the experimental and control groups at 30 and 45 dpi. Proteomic analysis identified 1660 and 1244 differentially expressed proteins (DEPs) at 30 and 40 dpi, respectively, compared with the uninfected spleen tissues. According to GO analysis, these DEPs were involved in processes such as organelle organization, cellular component biogenesis, cellular component assembly, anion binding, small molecule binding, metal ion binding, cation binding, cytosol, nuclear part, etc. Additionally, KEGG analysis indicated that the following pathways were linked to MDV-induced inflammation, apoptosis, and tumor: Wnt, Hippo, AMPK, cAMP, Notch, TGF-ß, PI3K-Akt, Rap1, Ras, Calcium, NF-κB, PPAR, cGMP-PKG, Apoptosis, VEGF, mTOR, FoxO, TNF, JAK-STAT, MAPK, Prion disease, T cell receptor, and B cell receptor. We finally screened 674 DEPs that were linked to MDV infection in spleen tissue. This study improves our understanding of the MDV response mechanism in the spleen.

Omicron breakthrough infections in wild-type SARS-CoV-2 vaccinees elicit high levels of neutralizing antibodies against pangolin coronavirus GX_P2V.

Omicron BF.7 became the predominant SARS-CoV-2 variant in Beijing after the abolishment of Zero-COVID policy in December 2022. The ability of antibodies elicited by BF.7 infection to cross-react with SARS-CoV-2-like viruses is unknown. This study aimed to investigate the cross-reactive neutralizing antibodies against SARS-CoV-2-related pangolin coronavirus GX_P2V in sera from vaccinated and/or SARS-CoV-2-infected individuals. All vaccinated individuals who recovered from Omicron BF.7 breakthrough infections exhibited substantially higher levels of neutralizing antibodies against GX_P2V, compared to other subject groups, with a geometric mean titer (GMT) of 362. Uninfected individuals who received four-mixed-dose vaccines also demonstrated higher levels of neutralizing antibodies (GMT = 44) against GX_P2V than those uninfected individuals who received two- or three-dose vaccines and those unvaccinated convalescents of wild-type SARS-CoV-2. This study highlights the significance of prior vaccinations with wild-type SARS-CoV-2 vaccines in generating potent cross-protective immunity against future spillovers of SARS-CoV-2-like viruses.

Adsorption of Cd (II) by a novel living and non-living Cupriavidus necator GX_5: optimization, equilibrium and kinetic studies.

Biosorbents have been extensively studied for heavy metal adsorption due to their advantages of low cost and high efficiency. In the study, the living and non-living biomass of Cupriavidus necator GX_5 previously isolated were evaluated for their adsorption capacity and/or removal efficiency for Cd (II) through batch experiments, SEM and FT-IR investigations. The maximum removal efficiency rates for the live and dead biomass were 60.51% and 78.53%, respectively, at an optimum pH of 6, a dosage of 1 g/L and an initial Cd (II) concentration of 5 mg/L. The pseudo-second-order kinetic model was more suitable for fitting the experimental data, indicating that the rate-limiting step might be chemisorption. The Freundlich isotherm model fit better than the Langmuir isotherm model, implying that the adsorption process of both biosorbents was heterogeneous. FT-IR observation reflected that various functional groups were involved in Cd (II) adsorption: -OH, -NH, C=O, C-O and C-C groups for the living biomass and -OH, -NH, C-H, C = O, C-N and N-H groups for the dead biomass. Our results imply that non-living biosorbents have a higher capacity and stronger strength for absorbing Cd (II) than living biomass. Therefore, we suggest that dead GX_5 is a promising adsorbent and can be used in Cd (II)-contaminated environments.

Three new lanostane triterpenoids and two new amides from Alternaria sp. with NLRP3 inflammasome inhibitory activity.

Three new lanostane triterpenoids (1-3) along with two new amides fatty compounds (4-5) were isolated from the ethyl acetate extract of a culture of the endophytic fungus Alternaria sp. gx-2. Their structures were identified by 1D and 2D NMR spectral data and HRESIMS. Compounds 1-12 were evaluated for their anti-inflammatory and tyrosinase inhibition activities. The isolated compounds did not show inhibitory activities at a concentration of 100 µM against tyrosinase, while under the concentration of 10 µM, the release of lactate dehydrogenase (LDH) inhibition rate of compound 1 was 54.45%, indicating that compound 1 had moderate anti-inflammatory activity on the activation of NLRP3 inflammasome.

An update on xylan structure, biosynthesis, and potential commercial applications.

•Xylan is an abundant carbohydrate component of plant cell walls that is vital for proper cell wall structure and vascular tissue development.•Xylan structure is known to vary between different tissues and species.•The role of xylan in the plant cell wall is to interact with cellulose, lignin, and hemicelluloses.•Xylan synthesis is directed by several types of Golgi-localized enzymes.•Xylan is being explored as an eco-friendly resource for diverse commercial applications.

A SARS-CoV-2-Related Virus from Malayan Pangolin Causes Lung Infection without Severe Disease in Human ACE2-Transgenic Mice.

Coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the most severe emerging infectious disease in the current century. The discovery of SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins in South Asian countries indicates that SARS-CoV-2 likely originated from wildlife. To date, two SARSr-CoV-2 strains have been isolated from pangolins seized in Guangxi and Guangdong by the customs agency of China, respectively. However, it remains unclear whether these viruses cause disease in animal models and whether they pose a transmission risk to humans. In this study, we investigated the biological features of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin (Manis javanica) captured by the Guangxi customs agency, termed MpCoV-GX, in terms of receptor usage, cell tropism, and pathogenicity in wild-type BALB/c mice, human angiotensin-converting enzyme 2 (ACE2)-transgenic mice, and human ACE2 knock-in mice. We found that MpCoV-GX can utilize ACE2 from humans, pangolins, civets, bats, pigs, and mice for cell entry and infect cell lines derived from humans, monkeys, bats, minks, and pigs. The virus could infect three mouse models but showed limited pathogenicity, with mild peribronchial and perivascular inflammatory cell infiltration observed in lungs. Our results suggest that this SARSr-CoV-2 virus from pangolins has the potential for interspecies infection, but its pathogenicity is mild in mice. Future surveillance among these wildlife hosts of SARSr-CoV-2 is needed to monitor variants that may have higher pathogenicity and higher spillover risk. IMPORTANCE SARS-CoV-2, which likely spilled over from wildlife, is the third highly pathogenic human coronavirus. Being highly transmissible, it is perpetuating a pandemic and continuously posing a severe threat to global public health. Several SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins have been identified since the SARS-CoV-2 outbreak. It is therefore important to assess their potential of crossing species barriers for better understanding of their risk of future emergence. In this work, we investigated the biological features and pathogenicity of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin, named MpCoV-GX. We found that MpCoV-GX can utilize ACE2 from 7 species for cell entry and infect cell lines derived from a variety of mammalian species. MpCoV-GX can infect mice expressing human ACE2 without causing severe disease. These findings suggest the potential of cross-species transmission of MpCoV-GX, and highlight the need of further surveillance of SARSr-CoV-2 in pangolins and other potential animal hosts.

Carboxymethyl chitosan-assisted MnOx nanoparticles: Synthesis, characterization, detection and cartilage repair in early osteoarthritis.

While the early detection and repair of cartilage lesions are crucial in the treatment of osteoarthritis (OA), they remain challenging because neither clinically used medicines nor magnetic resonance (MR) contrast agents can achieve detection and repair simultaneously. Here, we conjugated carboxymethyl chitosan (CMC) with a cartilage-targeting peptide (WYRGRL, termed WY) and then synthesized CMC-assisted manganese oxide nanoparticles (MnOx NPs). The resultant WY-CMC-MnOx NPs demonstrated an excellent biocompatibility and a good T1 relaxivity of 1.72 mM-1·s-1. Owing to their ultrasmall size and cartilage-targeting ability, the WY-CMC-MnOx NPs considerably increased the MR imaging quality of cartilage lesions compared to non-cartilage-targeting NPs. In contrast, clinically used gadolinium-diethylenetriamine pentaacetic acid (Gd-DPTA) failed to detect the cartilage lesions. Furthermore, WY-CMC-MnOx promoted chondrogenesis in mesenchymal stem cells, thereby enhancing OA therapy through efficient cartilage regeneration after intraarticularly injection in destabilization of medial meniscus (DMM) rat models. Our results indicate that WY-CMC-MnOx NPs are promising for use in the diagnosis and treatment of early OA.

Characteristics of blood plasma proteome changes associated with the hemorrhagic purpura of cosmonauts on the first day after long-term space missions.

The interest in the role of the gravitational factor during landing after long-term space flights (SF) leads to the search for various innovative approaches to assessing the compliance of external changes observed by clinicians. The results of special research methods such as Omics technologies that may reflect physiological responses to the conditions created during landing are of great interest. Our purpose is to compare the blood plasma proteome changes associated with the trauma and endothelial dysfunction processes prior to launch and on the day of landing, as well as the groups of cosmonauts with and without the secondary hemorrhagic purpura. In our study, the concentrations of 125 plasma proteins in 18 Russian cosmonauts, measured using targeted proteomic analysis based on liquid chromatography and tandem mass spectrometry were analyzed. The results reveal the trends of 12 proteins participating in the processes that trigger hemorrhagic purpura under the effect of re-entry g-forces. Exposure to intense g-forces and return to the gravity are the key factors for external manifestations of changes in the body systems induced by a long-term stay in space microgravity. Our results may be useful for further research to experts in gravitational physiology, aviation and space medicine.

Progress on the investigation and monitoring of marine phycotoxins in China.

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin ß-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus.

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

68Ga-Labeled GX1 Dimer: A Novel Probe for PET/Cerenkov Imaging Targeting Gastric Cancer.

PURPOSE: To synthesize the dimer of GX1 and identify whether its affinity and targeting are better than those of GX1. To prepare 68Ga-DOTA-KEK-(GX1)2 and to apply it to PET and Cerenkov imaging of gastric cancer. METHODS: 68Ga-DOTA-KEK-(GX1)2 was prepared, and the labeling yield and stability were determined. Its specificity and affinity were verified using an in vitro cell binding assay and competitive inhibition test, cell immunofluorescence, and cell uptake and efflux study. Its tumor-targeting ability was determined by nano PET/CT and Cerenkov imaging, standardized uptake value (SUV), signal-to-background ratio (SBR) quantification, and a biodistribution study in tumor-bearing nude mice. RESULTS: 68Ga-DOTA-KEK-(GX1)2 was successfully prepared, and the labeling yield was more than 97%. It existed stably for 90 min in serum. The binding of 68Ga-DOTA-KEK-(GX1)2 to cocultured HUVECs (Co-HUVECs) was higher than that to human umbilical vein endothelial cells (HUVECs), BGC823 cells, and GES cells. It was also higher than that of 68Ga-DOTA-GX1, indicating that the dimer did improve the specificity and affinity of GX1. The binding of KEK-(GX1)2 to Co-HUVECs was significantly higher than that of GX1. Additionally, the uptake of 68Ga-DOTA-KEK-(GX1)2 by Co-HUVECs was higher than that of 68Ga-DOTA-GX1 and reached a maximum at 60 min. Nano PET/CT and Cerenkov imaging showed that the tumor imaging of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 was clear, and the SUV and SBR value of the tumor sites were significantly higher than those of the nude mice injected with 68Ga-DOTA-GX1, indicating that the probe had better targeting in vivo. Finally, the biodistribution showed quantitatively that when organs such as the kidney and liver metabolized rapidly, the radioactivity of the tumor site of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 decreased relatively slowly. At the same time, the percentage of injected dose per gram (%ID/g) of the tumor site was higher than that of other normal organs except the liver and kidney at 60 min, which indicated that the tumor had good absorption of the probe. CONCLUSION: GX1 was modified successfully, and the in vivo and in vitro properties of the GX1 dimer were significantly better than those of GX1. The imaging probe, 68Ga-DOTA-KEK-(GX1)2, was successfully prepared, which provides a candidate probe for PET and Cerenkov diagnosis of gastric cancer.

The tumour neovasculature-homing dimeric peptide GX1 demonstrates antiangiogenic activity in the retinal neovasculature.

Identification of molecules specific to the retinal neovasculature will promote antiangiogenic therapy with enhanced targeting ability. The specificity of phage-displayed peptide GX1 (a cyclic 7-mer peptide motif CGNSNPKSC) to gastric cancer neovasculature has been extensively confirmed both in vitro and in vivo. To investigate the potential application of GX1 in antiangiogenic therapy targeting retinal angiogenesis-related diseases, we performed immunohistochemistry and immunofluorescence analyses. GX1 demonstrated positive staining in the retinal neovasculature in an oxygen-induced mouse model of retinopathy (OIR) as well as in rat retinal microvasculature endothelial cells (RMECs), confirming the major role of the GX1 receptor during retinal angiogenesis. Dimeric GX1 was synthesized to increase the binding affinity to the GX1 receptor, and the antiangiogenic effects were examined in RMECs in vitro and the retinal neovasculature in the OIR in vivo. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8) assay, revealing that compared with the GX1 monomer, dimeric GX1 significantly inhibited RMEC proliferation (P < 0.05). This finding may be attributed to the enhanced (P < 0.05) apoptosis induced by dimeric GX1 in RMECs based on results obtained from TUNEL, flow cytometric and cell cycle analyses. In RMECs, in vitro cell migration and tube formation were significantly inhibited following exposure to dimeric GX1. Intravitreal administration of dimeric GX1 resulted in a greater reduction in the retinal neovascularization in vivo than administration of the GX1 monomer (P < 0.05). In conclusion, dimeric GX1 showed greater inhibition of angiogenesis than monomeric GX1 and could be a promising agent for antiangiogenic therapy in retinal angiogenesis-related diseases.

Artemether, Artesunate, Arteannuin B, Echinatin, Licochalcone B and Andrographolide Effectively Inhibit SARS-CoV-2 and Related Viruses In Vitro.

Since the first reported case caused by the novel coronavirus SARS-CoV-2 infection in Wuhan, COVID-19 has caused serious deaths and an ongoing global pandemic, and it is still raging in more than 200 countries and regions around the world and many new variants have appeared in the process of continuous transmission. In the early stage of the epidemic prevention and control and clinical treatment, traditional Chinese medicine played a huge role in China. Here, we screened out six monomer compounds, including artemether, artesunate, arteannuin B, echinatin, licochalcone B and andrographolide, with excellent anti-SARS-CoV-2 and anti-GX_P2V activity from Anti-COVID-19 Traditional Chinese Medicine Compound Library containing 389 monomer compounds extracted from traditional Chinese medicine prescriptions "three formulas and three drugs". Our discovery preliminary proved the stage of action of those compounds against SARS-CoV-2 and provided inspiration for further research and clinical applications.

Telomerase-based GX301 cancer vaccine in patients with metastatic castration-resistant prostate cancer: a randomized phase II trial.

Debate is around the optimal immunization regimen for cancer vaccines since too intense vaccination schedules may exhaust reactive lymphocytes. GX301 is a telomerase-based cancer vaccine whose safety and immunological effects were tested in a phase I trial applying an eight administrations schedule. Main objective of this study was to comparatively analyse safety and immunological response to three GX301 regimens in metastatic castration-resistant prostate cancer patients with response/disease stability after docetaxel chemotherapy. This was a multicentre, randomized, parallel-group, open-label trial registered with EudraCT (2014-000095-26) and ClinicalTrials.gov (NCT02293707, 2014). Ninety-eight patients were randomized to receive either eight (regimen 1), four (regimen 2) or two (regimen 3) vaccine administrations. Sixty-three patients were assessable for the primary immunological end-point. Vaccine-specific immune responses were evaluated by intracellular staining for IFN, elispot and cytotoxic assay at 90 and 180 days from baseline. No major side effects were recorded. A 54% overall immune responder rate was observed with 95% of patients showing at least one vaccine-specific immune response. Rate of immunological responders and number of immunizations were proportionally related, suggesting superiority of regimens 1 and 2 over regimen 3. Overall survival did not differ among regimens in both immunological responders and non-responders and was inversely associated (P = 0.002) with increase in the number of circulating CD8 + T regulatory cells at 180 days. These data indicate that GX301 cancer vaccine is safe and immunogenic in metastatic castration-resistant prostate cancer patients. Schedules with high number of administrations should be preferred in future studies due to their better immunological outcome.

Cellulosimicrobium composti sp. nov., a thermophilic bacterium isolated from compost.

A Gram-stain-positive, yellow-pigmented, non-motile actinobacterial strain, designated as BIT-GX5T, was isolated from a sesame husks compost collected in Beijing, PR China. This bacterium was found to be able to grow in the temperature range from 16 to 50 °C and had an optimal growth temperature at 45 °C. Its taxonomic position was analysed using a polyphasic approach. The 16S rRNA gene sequence (1482 bp) of strain BIT-GX5T was most similar to Cellulosimicrobium funkei ATCC BAA-886T (99.45%), Cellulosimicrobium cellulans LMG 16121T (99.17%) and Cellulosimicrobium marinum RS-7-4T (98.75%). The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of five housekeeping genes (gyrB, rpoB, recA, atpD and trpB) and genome sequences, placed strain BIT-GX5T in a separate lineage among the genus Cellulosimicrobium within the family Promicromonosporaceae. The major polar lipids of strain BIT-GX5T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid and aminolipid. The major isoprenoid quinone was MK-9(H4), while the cell-wall sugars were galactose, rhamnose, glucose and mannose. The peptidoglycan type was A4α l-Lys-d-Ser-d-Asp. The major fatty acids were anteiso-C15:0 and iso-C15: 0, which were similar to other members in the genus Cellulosimicrobium. Results of in silico DNA-DNA hybridization and average nucleotide identity calculations plus physiological and biochemical tests exhibited the genotypic and phenotypic differentiation of strain BIT-GX5T from the other members of the genus Cellulosimicrobium. Therefore, strain BIT-GX5T is considered to represent a novel species within the genus Cellulosimicrobium, for which the name Cellulosimicrobium composti sp. nov. is proposed. The type strain is BIT-GX5T (= CGMCC 1.17687T = KCTC 49391T).

Identification of a novel protein in porcine astrovirus that is important for virus replication.

Overlapping genes are common in some RNA viruses. It has been proposed that a potential overlapping gene is the ORFX, here termed ORF2b, which overlaps the ORF2 coding sequence in astroviruses. The aim of this study was to determine whether ORF2b is an overlapping gene that encodes a functional protein which is needed for viral replication. Sequence alignment showed that there was an ORF2b in a PAstV type 1 strain of astrovirus, PAstV1-GX1, which was embedded within the larger ORF2. The AUG codon for ORF2b is located 19 nucleotides downstream of the initiation site of ORF2 and contains 369 nucleotides and it codes for a predicted 122-amino-acid protein. A specific polyclonal antibody against the ORF2b protein was raised and used to demonstrate the expression of the new identified gene in virus-infected and pCAGGS-ORF2b-transfected cells. Analysis of purified virions revealed that the ORF2b protein was not incorporated into virus particles. Reverse genetics based on a PAstV type 1 infectious cDNA clone showed that the ORF2b protein was not essential but important for optimal virus infectivity. Knockout of the downstream potential stop codon candidate of ORF2b demonstrated that the C-terminus of the ORF2b protein can be extended by 170 amino acids, suggesting that the C-terminus of the newly identified ORF2b protein may be variable.

Transcriptome analysis of cepharanthine against a SARS-CoV-2-related coronavirus.

Antiviral therapies targeting the pandemic coronavirus disease 2019 (COVID-19) are urgently required. We studied an already-approved botanical drug cepharanthine (CEP) in a cell culture model of GX_P2V, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related virus. RNA-sequencing results showed the virus perturbed the expression of multiple genes including those associated with cellular stress responses such as endoplasmic reticulum (ER) stress and heat shock factor 1 (HSF1)-mediated heat shock response, of which heat shock response-related genes and pathways were at the core. CEP was potent to reverse most dysregulated genes and pathways in infected cells including ER stress/unfolded protein response and HSF1-mediated heat shock response. Additionally, single-cell transcriptomes also confirmed that genes of cellular stress responses and autophagy pathways were enriched in several peripheral blood mononuclear cells populations from COVID-19 patients. In summary, this study uncovered the transcriptome of a SARS-CoV-2-related coronavirus infection model and anti-viral activities of CEP, providing evidence for CEP as a promising therapeutic option for SARS-CoV-2 infection.