Coxsackievirus B3 Infection Triggers Autophagy through 3 Pathways of Endoplasmic Reticulum Stress

Objective Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3(CVB3) infection induced autophagy through endoplasmic reticulum(ER) stress. Methods In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction(RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase(PERK) inhibitor, inositol-requiring protein-1(IRE1) inhibitor, or activating transcription factor-6(ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3(LC3). Results CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein(GFP)-LC3 punctuation and induced the conversion from LC3-Ⅰ to phosphatidylethanolamine-conjugated LC3-1(LC3-Ⅱ). CVB3 infection still decreased the expression of mammalian target of rapamycin(mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-Ⅱ to LC3-Ⅰ in CVB3-infected HeLa cells. Conclusion CVB3 infection induced autophagy through ER stress in HeL a cells, and PERK, IRE1, and ATF6 a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.

Leishmaniasis:Current status of available drugs and new potential drug targets

The control of Leishmania infection relies primarily on chemotherapy till date.Resistance to pentavalent antimonials,which have been the recommended drugs to treat cutaneous and visceral leishmaniasis,is now widespread in Indian subcontinents.New drug formulations like amphotericin B,its lipid formulations,and miltefosine have shown great efGcacy to treat leishmaniasis but their high cost and therapeutic complications limit their usefulness.In addition, irregular and inappropriate uses of these second line drugs in endemic regions like state of Bihar, India threaten resistance development in the parasite.In context to the limited drug options and unavailability of either preventive or prophylactic candidates,there is a pressing need to develop true antileishmanial drugs to reduce the disease burden of this debilitating endemic disease.Notwithstanding significant progress of leishmanial research during last few decades, identification and characterization of novel drugs and drug targets are far from satisfactory.This review will initially describe current drug regimens and later will provide an overview on few important biochemical and enzymatic machineries that could be utilized as putative drug targets for generation of true antileishmanial drugs.

Clinical Observation on Treatment of 2,062 Cases of Immune Infertility with Integration of Traditional Chinese Medicine and Western Medicine

To study the therapeutic effect of integrated traditional Chinese medicine and western medicine on female immune infertility. 3,496 women suffering from primary or secondary infertility had their ASAb, EMAb,AOAb and ACAb level tested, with the positive rate of 23.11%, 34.95%, 20.77% and 30.41% respectively.2,062 positive cases were periodically treated with the Chinese drug Xiaokangwan (消抗丸) plus dexamethasone, vitamin E and vitamin C for 2 periods as a course of treatment. At the end of a treatment course, the rate for the antibodies to turn negative reached over 85% and the average pregnant rate reached 36.66%. The treatment of immune infertility with the integrated approach can reduce or eliminate the influence of antibodies in the serum of patients on various links of pregnancy, thus reaching the goal of curing infertility.

Detection of urinary antigens and their seroreactivity with serum of patients in Leishmania donovani infection

Objective:To detect leishmanial antigens in pre and post treated urine of visceral leishmaniasis (VL) patients.Methods:Urine and serum sample from three VL patients were collected. Ammonium sulphate precipitation and purification of urine sample was done for proteins isolation.SDS PAGE of proteins was done followed by western blotting,with the patient’s pre and post treatment serum.Results:Eight proteins of molecular weights 17 kDa,25 kDa,28 kDa,42 kDa, 47 kDa,54 kDa,60 kDa and 85 kDa were detected in the urine of VL patients before treatment. After treatment with miltefosine,none of the above proteins was detected in urine samples.The western blot analysis with pre treatment serum confirmed the antigenicity of four urinary proteins of molecular weights 25 kDa,28 kDa,54 kDa and 60 kDa.The seropositivity with 25 kDa and 28 kDa antigens was negative with serum obtained after the completion of treatment.Conclusions:In the context to unavailability of a prognostic tool,urinary leishmanial antigens may offer a better choice and may also be useful as immunoprophylactic candidates.

Exploration of IRES Elements within the ORF of the Coxsackievirus B3 Genome

Objective This study aimed to identify internal ribosome entry sites(IRESs)in the open reading frame(ORF)of the Coxsackievirus B3(CVB3)genome.Methods The sequences of P1,P2,or P3 of the CVB3 genome or the truncated sequences from each antithymocyte globulin(ATG)to the end of the P1,P2,or P3 gene were inserted into the pEGFP-N1vector.After transfection,possible IRES-dependent green fluorescent protein(GFP)-fused proteins were detected by anti-GFP western blotting.The sequences of possible IRESs were inserted into specific Fluc/Rluc bicistronic vectors,in which the potential IRESs were determined according to the Fluc/Rluc activity ratio.Expression of Fluc and Rluc mRNA of the bicistronic vector was detected by RT-qPCR.Results After transfection of full length or truncated sequences of the P1,P2,or P3 plasmids,six GFPfused protein bands in P1,six bands in P2 and nine bands in P3 were detected through western blotting.Two IRESs in VP2(1461–1646 nt)and VP1(2784–2983 nt)of P1;one IRES in 2C(4119–4564 nt)of P2;and two IRESs in 3C(5634–5834 nt)and 3D(6870–7087 nt)of P3 were identified according to Fluc/Rluc activity ratio.The cryptic promoter was also excluded by RT-qPCR.Conclusion Five IRESs are present in the CVB3 coding region.

Dried blood spot sampling as an alternative for the improvement of hepatitis B and C diagnosis in key populations

BACKGROUND To achieve the elimination of hepatitis B and C,there is an urgent need to develop alternative strategies to increase the access of diagnosis,particularly among key populations such as people living with human immunodeficiency virus(HIV),individuals with coagulopathies and chronic kidney disease(CKD)patients.AIM To evaluate the use of dried blood spot(DBS)in the detection of hepatitis B virus(HBV)and hepatitis C virus(HCV)markers.METHODS A total of 430 individuals comprised of people living with HIV,coagulopathies and CKD provided paired serum and DBS samples.HBsAg,anti-HBc and anti-HCV were tested in those samples using a commercial electrochemiluminescence.Demographic and selected behavioral variables were evaluated to assess possible association with HBV and HCV positivity.RESULTS Using DBS,HBsAg prevalence varied from 3.9%to 22.1%,anti-HBc rates varied from 25.5%to 45.6%and anti-HCV positivity ranged from 15.9%to 41.2%in key populations.Specificities of HBV and HCV tests using DBS varied from 88.9%to 100%.The HBsAg assay demonstrated the best performance in CKD and coagulopathy individuals and the anti-HCV test had a sensitivity and specificity of 100%in people living with HIV.Accuracy of HBV and HCV detection in DBS varied from 90.2%to 100%.In the CKD group,HBsAg positivity was associated with infrequent use of condoms,and anti-HBc positivity was associated with sharing nail cutters/razors/toothbrushes.Anti-HCV reactivity was positively associated with a history of transplantation and length of time using hemodialysis in both specimens.In people living with HIV,only the male gender was associated with anti-HBc positivity in serum and DBS.CONCLUSION DBS with electrochemiluminescence are useful tools for the diagnosis and prevalence studies of hepatitis B and C among key populations and may increase the opportunity to foster prevention and treatment.

Atypical Hemolytic Uremic Syndrome in a Post-COVID-19 Child: Its Differential Diagnosis with COVID-19, Multisystemic Inflammatory Syndrome and Outcome

Pediatric Multisystemic Inflammatory Syndrome (MIS-C) is one of the most severe manifestations of SARS-CoV-2 in pediatrics [1]. This is a report of MIS-C with clinical presentation in infants with atypical Hemolytic Uremic Syndrome (aHUS).

Litopenaeus vannamei immunestimulated with Macrocystis pyrifera extract:improving the immune response against Vibrio campbellii

Objective:To examine the immune responses of Litopenaeus vannamei after different treatments with a hot water extract of Macrocystis pyrifera(M.pyrifera)and a subsequent challenge with Vibrio campbellii(V.campbellii).Methods:A total of 184 adult white shrimp that were infected with V.campbellii(1×10^(6) CFU/shrimp)were immunostimulate by the hot-water extract from M.pyrifera via either injection(10μg)or immersion(350 mg/L),the experimental controls were injected with either saline solution or V.campbellii(1×10^(6) CFU/shrimp).The bacterial DNA depuration rate,antimicrobial activity and total hemocyte count were evaluated in hemolymph samples at 2,6,12,24,48 and 72 h post-infection.Results:Injected shrimp(10μg M.pyrifera extract)demonstrated the best clearance of bacterial infection,with 82%survival at 72 h post-infection(cellular response).Hemolymph from the immersed organisms had the best antimicrobial activity against Escherichia coli growth;specifically,the most efficient antimicrobial activity was observed at 24 h post-infection.Both types of immunostimulated shrimp had similar total hemocyte counts at 24 h post-infection(1.63-1.59 million/mL);however,after 72 h,injected shrimp had higher total hemocyte counts than immersed animals(2.59 v.s.0.56 million/mL).Conclusions:The injection of the M.pyrifera hot-water extract facilitated a more efficient response to V.campbellii infection due to the stimulation of the hemocytes of the shrimp.In other words,the cellular immune response was more efficient to eliminate bacterial infection than the humoral response in shrimp.

Visual detection of tropomyosin,a major shrimp allergenic protein using gold nanoparticles(AuNPs)-assisted colorimetric aptasensor

A gold nanoparticle-based label-free colorimetric assay was developed to detect the shrimp allergenic protein tropomyosin(TM),an important biomarker responsible for severe clinical reactivity to shellfish.In a gold nanoparticles(AuNPs)-tropomyosin-binding aptamer(TMBA)complex,the aptamer adsorbs onto the surface of AuNPs and dissociates in the presence of TM.In addition,AuNPs tend to aggregate in the presence of ionic salt,revealing a color change(i.e.,wine-red to purple/blue)with a shift in the maximum absorption peak from 520 nm.In the presence of specific binding TM,the aptamer folds into a tertiary structure where it more efficiently stabilizes AuNPs toward the salt-induced aggregation with a hypsochromic shift in the absorption spectra compared to the stabilized AuNPs by aptamer alone.Based on the aggregation and sensitive spectral transformation principle,the AuNPs-based colorimetric aptasensor was successfully applied to detect TM with a range of 10-200 nmol/L and a low detection limit of 40 nmol/L in water samples.The reliability,selectivity,and sensitivity of the aptasensor was then tested with food samples spiked with TM.The observed detection limit was as low as 70 nmol/L in shrimp,90 nmol/L in tofu,and 80 nmol/L in eggs,respectively.We anticipate the proposed AuNPs-based colorimetric aptasensor assay possesses a high potential for the easy and efficient visual colorimetric detection of TM.

Progress in crystallization of major histocompatibility complex class I in vertebrates

The major histocompatibility complex(MHC)of proteins that exists in all vertebrates is encoded by a cluster of genes associated with the immune response and related functions.MHC is divided into MHC I,II,and III;MHC I is involved in antigenic presentation,binding T cell receptors,and leading ultimately to specific cellular immune responses.The complicated functions of MHC I are determined by the nature of the complex.The crystal structure of MHC I has been solved for many animals,revealing the relationship between spatial structure and function.MHC I consists of an a heavy chain and a b2m light chain,both ligated non-covalently to a complex when a peptide is bound to the antigenic-binding groove.The a heavy chain is divided into an extracellular domain,a transmembrane domain,and an intracellular domain.The extracellular domain consists of sub-regions a1,a2,and a3.The a1 and a2 together form the antigenic-binding groove and bind antigenic peptides with 8–10 amino acid residues.MHC I can form a stable spatial structure;however,it should be noted that there are differences in the structure of MHC I among animal species,including anchored amino acids in binding peptides,binding sites,molecular distance,crystallization conditions,etc.Here,progress in determination of the crystal structure of human,mouse,chicken,non-human primate,and swine MHC I is described in detail.