L1 Protein Research Articles

Design, Synthesis, and Evaluation of Peptides Derived from L1 Protein Against Bovine Papillomavirus-1/2 Identified Along Mexico's Cattle Export Route.

Bovine papillomatosis affects animal health and represents one of the greatest economic losses in the livestock sector. New control and prevention methods to protect the livestock industry from this disease are necessary. The aim of this study was to evaluate a candidate peptide for antibody production against bovine papillomavirus (BPV). A total of 64 cattle underwent wart excision among 5,485 cattle distributed over 2 to 4 farms per state and 12 farms in total in the four Mexican states of Tabasco, Chiapas, Veracruz, and Nuevo León. The prevalence of bovine papillomatosis per farm was calculated by wart visualisation. The warts were genotyped by PCR and sequenced, then a phylogenetic tree was built using MEGA X software. A synthetic peptide was designed in the ABCpred, Bepipred 2.0, Bepipred IDBT, Bepitope, LBtope, and MHC II predictor online server software's based on the C-terminal region of the L1 protein. Mice antibody production was induced by subcutaneous immunisation with 50 μg of synthetic peptide and evaluated by indirect ELISA. The prevalence of BPV was higher in Tabasco, Chiapas, and Veracruz. Bovine papillomaviruses 1 and 2 were found in all representative samples. A phylogenetic tree showed that Mexican sequences were located in exclusive clades yet were highly related to international ones. The peptide immunisation induced antibody titres of 1 : 10,000/1 : 1,000,000 against synthetic peptide and whole wart lysate (WWL), respectively. Co-infections of BPV-1 and -2 were found in all four states. Immunisation of BALB/C mice with BPV-1/2-derived synthetic peptide based on the C-terminal region of the major viral capsid protein L1 induced the production of specific antibodies able to recognise BPV-1/2 viral particles from bovine WWL.

Critical Residues Involved in the Coassembly of L1 and L2 Capsid Proteins of Human Papillomavirus 16.

Human papillomaviruses (HPV) are small DNA viruses associated with cervical cancer, warts, and other epithelial tumors. Structural studies have shown that the HPV capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers in a T=7 icosahedral lattice, coassembling with substoichiometric amounts of the minor capsid protein, L2. However, the residues involved in the coassembly of L1 and L2 remain undefined due to the lack of structure information. Here, we investigated the solvent accessibility surfaces (SASs) of the central cavity residues of the HPV16 L1 pentamer in the crystal structure because those internal exposed residues might mediate the association with L2. Twenty residues in L1 protein were selected to be analyzed, with four residues in the lumen of the L1 pentamer identified as important: F256, R315, Q317, and T340. Mutations to these four residues reduced the PsV (pseudovirus) infection capacity in 293FT cells, and mutations to R315, Q317, and T340 substantially perturb L2 from coassembling into L1 capsid. Compared with wild-type (WT) PsVs, these mutant PsVs also have a reduced ability to become internalized into host cells. Finally, we identified a stretch of negatively charged residues on L2 (amino acids [aa] 337 to 340 [EEIE]), mutations to which completely abrogate L2 assembly into L1 capsid and subsequently impair the endocytosis and infectivity of HPV16 PsVs. These findings shed light on the elusive coassembly between HPV L1 and L2. IMPORTANCE Over 200 types of HPV have been isolated, with several high-risk types correlated with the occurrence of cervical cancer. The HPV major capsid protein, L1, assembles into a T=7 icosahedral viral shell, and associates with the minor capsid protein, L2, which plays a critical role in the HPV life cycle. Despite the important role of the L2 protein, its structure and coassembly with L1 remain elusive. In this study, we analyzed the amino acid residues at the proposed interface between L1 and L2. Certain mutations at these sites decreased the amount of L2 protein assembled into the capsid, which, in turn, led to a decrease in viral infectivity. Knowledge about these residues and the coassembly of L1 and L2 could help to expand our understanding of HPV biology and aid in the development of countermeasures against a wide range of HPV types by targeting the L2 protein.

A CRR2-Dependent sRNA Sequence Supports Papillomavirus Vaccine Expression in Tobacco Chloroplasts

Human papillomavirus (HPV) infection is the leading cause of cervical cancer, and vaccination with HPV L1 capsid proteins has been successful in controlling it. However, vaccination coverage is not universal, particularly in developing countries, where 80% of all cervical cancer cases occur. Cost-effective vaccination could be achieved by expressing the L1 protein in plants. Various efforts have been made to produce the L1 protein in plants, including attempts to express it in chloroplasts for high-yield performance. However, manipulating chloroplast gene expression requires complex and difficult-to-control expression elements. In recent years, a family of nuclear-encoded, chloroplast-targeted RNA-binding proteins, the pentatricopeptide repeat (PPR) proteins, were described as key regulators of chloroplast gene expression. For example, PPR proteins are used by plants to stabilize and translate chloroplast mRNAs. The objective is to demonstrate that a PPR target site can be used to drive HPV L1 expression in chloroplasts. To test our hypothesis, we used biolistic chloroplast transformation to establish tobacco lines that express two variants of the HPV L1 protein under the control of the target site of the PPR protein CHLORORESPIRATORY REDUCTION2 (CRR2). The transgenes were inserted into a dicistronic operon driven by the plastid rRNA promoter. To determine the effectiveness of the PPR target site for the expression of the HPV L1 protein in the chloroplasts, we analyzed the accumulation of the transgenic mRNA and its processing, as well as the accumulation of the L1 protein in the transgenic lines. We established homoplastomic lines carrying either the HPV18 L1 protein or an HPV16B Enterotoxin::L1 fusion protein. The latter line showed severe growth retardation and pigment loss, suggesting that the fusion protein is toxic to the chloroplasts. Despite the presence of dicistronic mRNAs, we observed very little accumulation of monocistronic transgenic mRNA and no significant increase in CRR2-associated small RNAs. Although both lines expressed the L1 protein, quantification using an external standard suggested that the amounts were low. Our results suggest that PPR binding sites can be used to drive vaccine expression in plant chloroplasts; however, the factors that modulate the effectiveness of target gene expression remain unclear. The identification of dozens of PPR binding sites through small RNA sequencing expands the set of expression elements available for high-value protein production in chloroplasts.

Abstract 17: Ultra-early Activation Of Neuroinflammatory Cascade Following Acute Stroke: Plasma And Extracellular Vesicle Biomarkers From A Mobile Stroke Unit

Background: Neuroinflammation is ubiquitous in acute stroke and worsens outcome. However, the precise timing of the inflammatory response is unknown, hindering the design of acute anti-inflammatory therapeutic interventions. We used a mobile stroke unit (MSU) to identify the onset of the neuroinflammatory cascade within minutes of symptom onset. Methods: The study is a prospective, cohort investigation of ultra-early blood- and extracellular vesicle (EV)-derived markers of neuroinflammation and outcome in acute stroke. Blood was obtained on the MSU with head CT, and from healthy controls. Plasma biomarkers were analyzed with ELISA, SIMOA and ECL. EVs were isolated with HSP pull down. Confirmed strokes, mimics and healthy controls were compared. Variables included demographics, mRS, NIHSS and discharge disposition. Results: Forty one adults were analyzed, including 15 patients treated on the MSU between August 2021 and April 2022, and 26 controls. For MSU patients, median age was 74 (range 36-97) years, 60% were female, and 80% white. Ten (67%) were diagnosed as stroke, with 8 (53%) confirmed and 2 likely TIA or stroke averted by thrombolysis; 5 were stroke mimics. For strokes, median initial NIHSS score was 11 (range 4-19) and 6 (75%) received tPA. Blood was obtained a median of 58 (range 36-133) minutes after symptom onset. Within 36 minutes after stroke, plasma interleukin-6 (IL-6), neurofilament light chain (NfL), ubiquitin C-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) were elevated by as much as 10 times normal. In EVs, matrix metalloproteinase-9 (MMP-9), chemokine (C-X-C motif) ligand 4 (CXCL4), C-reactive protein (CRP), IL-6, osteopontin (OPN) and platelet and endothelial cell adhesion molecule 1 (PECAM1) were elevated. Inflammatory markers increased rapidly in the first two hours, and continued rising for 24 hours. Conclusions: This study found the inflammatory cascade is activated as early as 36 minutes after stroke, and progresses rapidly. This is earlier than observed previously in humans, and suggests injury from neuroinflammation may occur faster than had been surmised. The findings may inform development of immunomodulatory therapies for acute stroke, and lead to new diagnostic tools and improved outcomes.

Decipher potential biomarkers of diagnosis and disease activity for NMOSD with AQP4 using LC-MS/MS and Simoa

BackgroundNeuromyelitis optica spectrum disorders (NMOSD) is an autoimmune demyelinating disease, leading recurrently relapses and severe disability. There is a need for new biomarkers to meet clinical needs in diagnosis and monitoring. MethodsThrough liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis, brain lesions from NMO animal models were analyzed to identify potential biomarkers. Then, we assessed the levels of serum glial fibrillary acidic protein (sGFAP), neurofilament light chain (sNfL), Tau protein (sTau) and Ubiquitin C-terminal hydrolase L1 (sUCHL1) using an ultrasensitive single molecule array (Simoa) of AQP4-IgG + NMOSD patients, myelin oligodendrocyte glycoprotein antibody-associated disorder (MOGAD) patients, multiple sclerosis (MS) patients and healthy controls (HCs). Additionally, we further explored the early diagnosis value of these proteins. ResultsThere were 72 differentially expressed proteins between the NMO and control groups. NfL abundance was elevated when GFAP, UCHL1, and Tau abundance was decreased in the NMO group. Then, we observed that the sGFAP and sUCHL1 levels in patients with NMOSD in the early stage were significantly increased compared to those in control participants. Combined ROCs of the sGFAP, sNfL, and sUCHL1 levels to better predict NMOSD with relapse stages was optimal. Notably, univariate and multivariate analyses demonstrated that the sGFAP and sNfL levels were higher in patients with brain lesions, while the sUCHL1 levels were higher in those with spinal cord lesions during recent relapse. ConclusionsThese findings suggested that sGFAP, sNfL, and sUCHL1 displayed good diagnostic performance in AQP4-IgG + NMOSD and could be novel candidates for early discrimination.

Impact of retrotransposon protein L1 ORF1p expression on oncogenic pathways in hepatocellular carcinoma: the role of cytoplasmic PIN1 upregulation

BackgroundMolecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood.MethodsWe analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems.ResultsWe observed positive associations between L1 and activated TGFβ-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFβ-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFβ-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p.DiscussionOur data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue.

Master mitotic kinases regulate viral genome delivery during papillomavirus cell entry

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2’s chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.

A novel C-terminal modification method enhanced the yield of human papillomavirus L1 or chimeric L1-L2 virus-like particles in the baculovirus system.

Human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs) produced in the baculovirus system showed excellent safety and immunogenicity, but the relatively high production cost stands as a substantial barrier to extensive commercialization, especially in producing multivalent vaccines. Here, a novel method, C-terminal basic amino acid (aa) substitution, was developed for increasing VLP and chimeric VLP (cVLP) production in this system. A series of mutants of five HPV types, including three L1 VLPs (6L1, 11L1, and 52L1) and two L1-L2 cVLPs (16L1-33L2, 58L1-16L2), were constructed. We found that most mutants exhibited higher protein expression in Sf9 cells, among which the yields of the superior mutants, 6L1CS4, 11L1CS3, 52L1m4∆N13CS1, 16L1-33L2 CS1, and 58L1-16L2 CS3, were up to 40, 35, 20, 35, and 60mg/L, which respectively increased by 4.2-, 7.3-, 5-, 2.5-, and 3.4-fold, and they also showed robust immunogenicity and great stabilities. Additionally, we found that the increased level of steady-state mRNA may play a crucial role in promoting L1 protein expression. Our results demonstrated that this novel method was cost-effective and can be used to reduce the production costs of L1 VLPs and L1-L2 cVLPs to develop broadly protective and affordable multivalent HPV vaccines.

Assessment of Risk Factors for Postoperative Delirium in Older Adults Who Underwent Spinal Surgery and Identifying Associated Biomarkers Using Exosomal Protein.

With an increase in the aging population, the number of patients with degenerative spinal diseases undergoing surgery has risen, as has the incidence of postoperative delirium. This study aimed to investigate the risk factors affecting postoperative delirium in older adults who had undergone spine surgery and to identify the associated biomarkers. This study is a prospective study. Data of 100 patients aged ≥ 70 years who underwent spinal surgery were analyzed. Demographic data, medical history, clinical characteristics, cognitive function, depression symptoms, functional status, frailty, and nutritional status were investigated to identify the risk factors for delirium. The Confusion Assessment Method, Delirium Rating Scale-R-98, and Nursing Delirium Scale were also used for diagnosing delirium. To discover the biomarkers, urine extracellular vesicles (EVs) were analyzed for tau, ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), neurofilament light, and glial fibrillary acidic protein using digital immunoassay technology. Nine patients were excluded, and data obtained from the remaining 91 were analyzed. Among them, 18 (19.8%) developed delirium. Differences were observed between participants with and without delirium in the contexts of a history of mental disorder and use of benzodiazepines (p = .005 and p = .026, respectively). Tau and UCH-L1-concentrations of urine EVs-were comparatively higher in participants with severe delirium than that in participants without delirium (p = .002 and p = .001, respectively). These findings can assist clinicians in accurately identifying the risk factors before surgery, classifying high-risk patients, and predicting and detecting delirium in older patients. Moreover, urine EV analysis revealed that postoperative delirium following spinal surgery is most likely associated with brain damage.

Mutation Profile of HPV16 L1 and L2 Genes in Different Geographic Areas

The causal relationship between HPV and cervical cancer in association with the high prevalence of high risk HPV genotypes led to the design of HPV vaccines based on the major capsid L1 protein. In recent years, capsid protein L2 has also become a focal point in the field of vaccine research. The present review focuses on the variability of HPV16 L1 and L2 genes, emphasizing the distribution of specific amino acid changes in the epitopes of capsid proteins. Moreover, a substantial bioinformatics analysis was conducted to describe the worldwide distribution of amino acid substitutions throughout HPV16 L1, L2 proteins. Five amino acid changes (T176N, N181T; EF loop), (T266A; FG loop), (T353P, T389S; HI loop) are frequently observed in the L1 hypervariable surface loops, while two amino acid substitutions (D43E, S122P) are adjacent to L2 specific epitopes. These changes have a high prevalence in certain geographic regions. The present review suggests that the extensive analysis of the amino acid substitutions in the HPV16 L1 immunodominant loops may provide insights concerning the ability of the virus in evading host immune response in certain populations. The genetic variability of the HPV16 L1 and L2 epitopes should be extensively analyzed in a given population.

Identification of diverse papillomaviruses in captive black-and-white ruffed lemurs (Varecia variegata).

Papillomaviruses (PVs) are host-species-specific and tissue-specific viruses that infect a diverse array of vertebrate hosts, including humans and non-human primates, with varying pathogenic outcomes. Although primate PVs have been studied extensively, no complete genome sequences of PVs from lemurs have been determined to date. Saliva samples from three critically endangered, captive black-and-white ruffed lemurs (Varecia variegata variegata) at the Duke Lemur Center (USA) were analyzed, using high-throughput sequencing, for the presence of oral papillomaviruses. We identified three PVs from two individuals, one of which had a coinfection with two different PVs. Two of the three PVs share 99.6% nucleotide sequence identity, and we have named these isolates "Varecia variegata papillomavirus 1" (VavPV1). The third PV shares ~63% nucleotide sequence identity with VavPV1, and thus, we have named it "Varecia variegata papillomavirus 2" (VavPV2). Based on their E1+E2+L1 protein sequence phylogeny, the VavPVs form a distinct clade. This clade likely represents a novel genus, with VavPV1 and VavPV2 belonging to two distinct species. Our findings represent the first complete genome sequences of PVs found in lemuriform primates, with their presence suggesting the potential existence of diverse PVs across the over 100 species of lemurs.

B-cell Epitope Mapping of Capsid L1 from Human Papillomavirus to Development Cervical Cancer Vaccine Through In Silico Study

Human papillomavirus (HPV) is a virus that plays an important role in the occurrence of cervical cancer. The HPV gene is composed of two parts: early and late gene. The L1 protein has a conserved region composed of cysteine ​​and lysine residues, both of which have involved in the binding process between virions and host receptors. Previous research has shown that vaccines can be developed based on epitopes that have conserved areas. This study is important to identify conserved protein sequences in L1 of HPV capsid, predict epitope mapping of B cells and antigenicity in the conserved region of L1 HPV capsid, as well as the similarity of amino acid residues of epitope composers with surface receptors of human body cells. The conserved areas were identified in L1 HPV as a potential epitope of B cells based on epitope mapping analysis of positions 23-46 and 97-119 with EGRGQPLGGSGHPNDDE DRDKQ and RHNGGPGPSGSSQFNKPYWAQGN peptides and each had a peptide length of 22-mer and 23-mer. The 97-119 epitope has a high antigenicity score and the similarity of the low amino acid residue sequence to the cell surface receptor of the human body, the 23-mer RHNGGPGPSGSSQFNKPYWAQGN peptide can be used as a reference for the development of cervical cancer prevention vaccine.

High relatedness of bioinformatic data and realistic experimental works on the potentials of Fasciola hepatica and F. gigantica cathepsin L1 as a diagnostic and vaccine antigen.

Fascioliasis is a parasitic foodborne disease caused by the liver flukes, Fasciola hepatica and F. gigantica. Such parasites cause serious illness in numerous domestic animals and also in humans. Following infection, the parasite secretes a variety of molecules that immediately interact with the host immunity to establish successful infection. These molecules include cathepsin L peptidase 1 (CatL1); the highly investigated diagnostic and vaccine antigens using various animal models. However, a few studies have analyzed the potentials of FhCatL1 as a diagnostic or vaccine antigen using bioinformatic tools and much less for FgCatL1. The present study provides inclusive and exclusive information on the physico-chemical, antigenic and immunogenic properties of F. hepatica cathepsin L1 (FhCatL1) protein using multiple bioinformatic analysis tools and several online web servers. Also, the validation of our employed available online servers was conducted against a huge collection of previously published studies focusing on the properties of FhCatL1as a diagnostic and vaccine antigen. For this purpose, the secondary, tertiary, and quaternary structure of FhCatL1 protein were also predicted and analyzed using the SWISS-MODEL server. Validation of the modeled structures was performed by Ramachandran plots. The antigenic epitopes of the protein were predicted by IEDB server. Our findings revealed the low similarity of FhCatL1 with mammalian CatL1, lacking signal peptides or transmembrane domain, and the presence of 33 phosphorylation sites. Also, the containment of FhCatL1 for many topological, physico-chemical, immunological properties that favored its function of solubility and interaction with the immune components were reported. In addition, the earlier worldwide reports documented the high efficacy of FhCatL1 as a diagnostic and vaccine antigen in different animals. Altogether, FhCatL1 is considered an excellent candidate for using in commercialized diagnostic assays or vaccine products against fascioliasis in different animal species. Our assessment also included FgCatL1 and reported very similar findings and outputs to those of FhCatL1.

Effects of Physical Exertion on Early Changes in Blood-Based Brain Biomarkers: Implications for the Acute Point of Care Diagnosis of Concussion.

Blood-based brain biomarkers (BBM) such as glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have potential to aid in the diagnosis of concussion. Recently developed point-of-care test devices would enable BBMs to be measured in field settings such military and sport environments within minutes of a suspicious head hit. However, head hits in these environments typically occur in the setting of vigorous physical exertion, which can itself increase BBMs levels. Thus, efforts to develop BBMs as acute concussion aids in field settings need to account for the effects of physical exertion. To determine the acute effects of physical exertion on the BBMs, we measured GFAP, UCH-L1, tau, and neurofilament light chain (NF-L) immediately before, immediately after, and 45 min after a single workout session consisting of aerobic and resistance exercises in 30 collegiate football players. Subjects wore body sensors measuring several aspects of exertion and underwent diffusion tensor imaging 24 h before and 48 h after exertion. All subjects were male with a mean age of 19.5 ± 1.2 years. The mean duration of activity during the workout session was 94 ± 31 min. There was a significant decrease in serum GFAP immediately after (median decrease of 27.76%, p < 0.0001) and a significant increase in serum UCH-L1 45 min after (median increase of 37.11%, p = 0.016) exertion, compared with pre-exertion baseline. No significant changes in tau or NF-L were identified. The duration of exertion had a significant independent linear correlation to the increase in serum UCHL1 from pre-exertion to 45 min after exertion (r = 0.68, p = 0.004). There were no significant pre- to post-exertional changes in any of the 39 examined brain white matter regions, and biomarker changes did not correlate to variation in white matter integrity in any of these regions. Thus, exertion appeared to be associated with immediate decreases in serum GFAP and very acute (45 min) increases in UCH-L1. These changes were related to the duration of exertion, but not to changes in brain white matter integrity. Our results have important implications for how these BBMs might be used to aid in the on-scene diagnosis of concussion occurring in the setting of physical exertion.

Abstract 265: Social Determinants Of Health Impact Phenotype-based Outcome Prediction After Pediatric Arrest

The Personalizing Outcomes after Child Cardiac Arrest-1 (POCCA-1) trial showed that blood-based brain injury biomarkers have promise to prognosticate outcome post-pediatric cardiac arrest. However, whether POCCA-1 biomarkers perform accurately among all children is unknown. Hypothesis We hypothesized that POCCA-1 biomarkers will have different performance accuracy by distinct clinical phenotypes after pediatric cardiac arrest. Methods: Fourteen US centers enrolled 164 children aged &lt; 18 y with pediatric intensive care unit admission post-arrest. Glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal esterase L1, neurofilament light, and Tau proteins were measured in blood post-arrest d1-3. Patient ZIP codes linked to US census data provided socioeconomic neighborhood characteristics. We analyzed 120 children with evaluable data (n=50, unfavorable outcome [Vineland Adaptive Behavioral Scale &lt; 70 or dead]) to create 3 clinical phenotypes using latent class analysis. POCCA-1 biomarker levels and outcomes were analyzed by phenotype. The POCCA-1 model was analyzed to predict 1 y unfavorable outcome using multivariate AUROC by phenotype for all 4 biomarkers on d1- 3 post-arrest. Results: Group 1 (n=35, 22%) were older, of White race, with private insurance and out-of-hospital cardiac arrests (OOHCA), fewer fever post-arrest, living in neighborhoods of mostly White race and lower poverty. Group 2 patients (n=77, 47%) were younger, of White race, with in-hospital cardiac arrests living in more socioeconomically diverse neighborhoods. Group 3 (n=51, 31%) were diverse in race and age, of more male sex, with Medicaid insurance and asphyxial, OOHCA, living in more impoverished neighborhoods. Unfavorable outcome was higher in Group 3 (69%) vs 1 (48%) and 2 (28%), p&lt;0.001. All biomarkers (except Tau) were significantly increased on d1-3 in Group 3. Biomarker accuracy to predict outcome was excellent for Group 1 with 11/12 AUROCs significant. Models were less accurate for Group 2 (7/12) and marginal for Group 3 (3/12). Conclusions: The POCCA-1 prediction model performance varied by patient, arrest, and neighborhood context, suggesting that prediction models need to be personalized using clinically relevant and equitable phenotypes.

Serologic Response to Human Papillomavirus Genotypes Among Unvaccinated Women: Findings From the HITCH Cohort Study.

Humoral immune responses may be critical for preventing, controlling, and/or eliminating human papillomavirus (HPV) infection. We analyzed humoral response to natural HPV infection considering phylogenetic relatedness among unvaccinated women. We included 399 young women attending university/college in Montreal, Canada who were participants of the HITCH cohort. Participants provided blood samples at baseline and 5 follow-up visits. Antibody response to bacterially expressed L1 and E6 glutathione S-transferase (GST) fusion proteins, and virus-like particles (VLP-L1) of Alphapapillomavirus types were measured using multiplex serology. We assessed correlations and associations between HPV types at baseline using Pearson correlation coefficients (r) and univariable linear regressions. At baseline, > 40% were seropositive for GST-L1 antibodies of at least 1 HPV type. Strong correlations between GST-L1 were observed for α9 HPV types: 58-52 (r = 0.86), 58-33 (r = 0.75), 33-52 (r = 0.72), and between GST-E6: 52-11 (r = 0.84), 52-18 (r = 0.79), 58-33 (r = 0.78), 35-11 (r = 0.76). HPV16 VLP-L1 moderately explained variability in HPV16 GST-L1 (regression coefficient [b] = 0.38, R2 = 43.1%), and HPV45 GST-L1 in HPV18 GST-L1 (b = 0.68, R2 = 42.8%). GST-E6 antibodies accounted for a low to moderate proportion of variability in HPV16 and HPV18 GST-E6 (R2 = 6.4%-62.2%). Associations between naturally induced HPV-specific antibodies depend on phylogenetic relatedness.

Characterization of an Escherichia coli-derived triple-type chimeric vaccine against human papillomavirus types 39, 68 and 70

In vaccinology, a potent immunogen has two prerequisite attributes—antigenicity and immunogenicity. We have rational designed a triple-type HPV vaccine against HPV58, −33 and −52 covered in Gardasil 9 based on the sequence homology and similar surface loop structure of L1 protein, which is related to cross-type antigenicity. Here, we design another triple-type vaccine against non-vaccine types HPV39, −68 and −70 by immunogenicity optimization considering type specific immunodominant epitopes located in separate region for different types. First, we optimized the expression of wild-type HPV39, −68 and −70 L1-only virus-like particles (VLPs) in E. coli through N-terminal truncation of HPV L1 proteins and non-fusion soluble expression. Second, based on genetic relationships and an L1 homologous loop-swapping rationale, we constructed several triple-type chimeric VLPs for HPV39, −68 and −70, and obtained the lead candidate named H39–68FG-70DE by the immunogenicity optimization using reactivity profile of a panel type-specific monoclonal antibodies. Through comprehensive characterization using various biochemical, VLP-based analyses and immune assays, we show that H39–68FG-70DE assumes similar particulate properties as that of its parental VLPs, along with comparable neutralization immunogenicity for all three HPV types. Overall, this study shows the promise and translatability of an HPV39/68/70 triple-type vaccine, and the possibility of expanding the type-coverage of current HPV vaccines. Our study further expanded the essential criteria on the rational design of a cross-type vaccine, i.e. separate sites with inter-type similar sequence and structure as well as type-specific immunodominant epitope to be clustered together.

Association between Brain Injury Markers and Testosterone in Critically-Ill COVID-19 Male Patients.

Accumulating data suggest that various neurologic manifestations are reported in critically-ill COVID-19 patients. Although low testosterone levels were associated with poor outcomes, the relationship between testosterone levels and indices of brain injury are still poorly understood. Therefore, we aimed to explore whether testosterone levels are associated with glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), biomarkers of brain injury, in patients with a severe form of COVID-19. The present study was conducted on 65 male patients aged 18-65 with severe COVID-19. Blood samples were collected at three time points: upon admission to ICU, 7 days after, and 14 days after. In patients with neurological sequels (n = 20), UCH-L1 serum concentrations at admission were markedly higher than in patients without them (240.0 (155.4-366.4) vs. 146.4 (92.5-243.9) pg/mL, p = 0.022). GFAP concentrations on admission did not differ between the groups (32.2 (24.2-40.1) vs. 29.8 (21.8-39.4) pg/mL, p = 0.372). Unlike GFAP, UCH-L1 serum concentrations exhibited a negative correlation with serum testosterone in all three time points (r = -0.452, p &lt; 0.001; r = -0.430, p &lt; 0.001 and r = -0.476, p = 0.001, respectively). The present study suggests that the traumatic brain injury biomarker UCH-L1 may be associated with neurological impairments seen in severe COVID-19. Moreover, a negative correlation between UCH-L1 and serum testosterone concentrations implies that testosterone may have a role in the development of neurological sequels in critically-ill COVID-19 patients.

Umbilical Cord-Derived Mesenchymal Stem Cells for the Treatment of Infertility Due to Premature Ovarian Failure.

Females belonging to the reproductive age group may face challenges regarding infertility or miscarriage due to conditions such as premature ovarian failure (POF). It is the conditionthat happens when a female's ovaries stop working before she is 40. The majority of the causes of POF cases are idiopathic. Other reasons includegenetic disorders (Turner's syndrome, bone morphogenetic protein 15 (BMP15) mutation, galactosemia, mutation of forkhead box protein L2 (FOXL2), growth differentiation factor-9 (GDF9), mutation of luteinizing hormone (LH) and follicle-stimulating hormonereceptors (FSHR), etc.), enzymatic mutation such as aromatase, autoimmune disorders (Addison's disease, vitiligo, systemic lupus erythematosus, myasthenia gravis, autoimmune thyroiditis, autoimmune polyglandular syndrome, etc.), vaccination, and environmental factors (cigarette smoking, toxins, and infections). Manyattempts have been made to treat POF by various methods. Some of the methods of treatment include hormone replacement therapy (HRT), melatonin therapy, dehydroepiandrosterone (DHEA) therapy, and stem cell therapy. Stem cell therapy has proven to be the most efficient form for treating POF as compared to all other options. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are the best among the other sources of mesenchymal stem cells (MSCs) for the treatment of POF as they have a painless extraction procedure. They have a tremendous capacity for self-repair and regeneration, which helps them in restoring degenerated ovaries. This review includes informationon the causes of POF, its efficacious therapeutic approaches, and the impact of transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs) as an option for the therapy of POF. Numerous studies conducted on stem cell therapy provethat it is an effective approach for the treatment of sterility.

In Vivo Capsid Engineering of Bacteriophages for Oriented Surface Conjugation.

The current state-of-the-art in bacteriophage (phage) immobilization onto magnetic particles is limited to techniques that are less expensive and/or facile but nonspecific or those that are more expensive and/or complicated but ensure capsid-down orientation of the phages, as necessary to preserve infectivity and performance in subsequent applications (e.g., therapeutics, detection). These cost, complexity, and effectiveness limitations constitute the major hurdles that limit the scale-up of phage-based strategies and thus their accessibility in low-resource settings. Here, we report a plasmid-based technique that incorporates a silica-binding protein, L2, into the T7 phage capsid, during viral assembly, with and without inclusion of a flexible linker peptide, allowing for targeted binding of the phage capsid to silica without requiring the direct modification of the phage genome. L2-tagged phages were then immobilized onto silica-coated magnetic nanoparticles. Inclusion of the flexible linker between the phage capsid protein and the L2 protein improved immobilization density compared to both wild type T7 phages and L2-tagged phages without the flexible linker. Taken together, this work demonstrates phage capsid modification without engineering the phage genome, which provides an important step toward reducing the cost and increasing the specificity/directionality of phage immobilization methods and could be more broadly applied in the future for other phages for a range of other capsid tags and nanomaterials.