Objective To develop a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of residual phenylmethanesulfonyl fluoride(PMSF) content in recombinant virus-like particle(VLP) vaccine stock solution,and to verify the method for the determination of PMSF residues in recombinant VLP vaccine stock solution.Methods A RP-HPLC method for the determination of PMSF residues was developed by screening detection wavelength,flow rate,injection amount and chromatographic column.The specificity,linearity and range,limit of quantitation(LOQ),limit of detection(LOD),accuracy,precision and durability of the method were verified.The developed method was used to detect PMSF residues in three batches of recombinant hepatitis E vaccine stock solution.Results The RP-HPLC method was developed,with ChromCore 300 C18(4.6 mm × 250 mm,5 μm) as the chromatographic column at the column temperature of 25 ℃,the detection wavelength of 210 nm,the injection amount of 100 μL,0.1% trifluoroacetic acid(TFA)/water and0.1% TFA/acetonitrile as mobile phase at a flow rate of 1 mL/min,and the detection time of 18 min.The absorption peaks of PMSF control and vaccine stock solution containing PMSF appeared around 5.0 min,while the vaccine stock solution and stock solution buffer showed no absorption peak.There was a good linear relationship between the concentration of PMSF control and peak area in the range of 0-5.0 μg/mL,with R~2 of 0.999.The LOQ and LOD of this method was 0.250 μg/mL and 0.125 μg/mL,respectively.The recovery rates of PMSF control with high,medium and low concentrations were all between 90% and 110%.The relative standard deviations(RSDs) of retention time,peak height and peak area for the determination of instrument repeatability,sample repeatability and intermediate precision were all less than 5%.The RSDs of retention time,peak height and peak area were all less than 5% under the TFA concentration of 0.09%,0.1% and 0.11% at column temperature of 20,25 and 30 ℃.PMSF was not detected in the three batches of recombinant hepatitis E vaccine stock solution.Conclusion The developed RP-HPLC method has good specificity,linearity and durability,high accuracy and precision,and can be used to detect residual PMSF content in recombinant VLP vaccine stock solution.
Phenylmethanesulfonyl fluoride(PMSF) ; Reversed-phase high-performance liquid chromatography(RP-HPLC) ; Virus-like particle(VLP) vaccine

Cough is a very common symptom causing medical consult. Several remedies are readily available in the market however these are currently not recommended among the pediatric population due to a few reasons which include the benign nature of acute cough, limited effectivity and lack of support from the United States Food and Drug Administration (USFDA) due to abuse potential.

We report a case of a 2-year-old male, no known co-morbidities with a 2 week history of upper respiratory tract infection. Initial assessment showed viral infection hence patient was given medications for symptomatic treatment. However, l week after, patient still presented with symptomatic persistent coughing that disrupted his activities of daily living, hence antitussive medication was already prescribed. After another 7 days, there was still persistence of symptoms, hence patient was given a trial medication of Pregabalin 0.7 milligram/kg/dose which noted instant cough relief one hour after the initial intake. Patient also reported to be more playful, improved sleep at night and improved appetite. Patient received total of 2 doses of Pregabalin in the span of 48 hours. On the third day, patient was still coughing but reported to be significantly less frequent and more productive, hence medication was then put on hold. Patient continuously improved after 5 more days and was eventually cough free.

This case report demonstrates the adequacy of Pregabalin as a supportive antitussive medication in a patient with an acute cough secondary to a viral infection.

Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×10⁴ were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 10⁵, and 3D5B7 reached 10⁷. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.
Horses ; Animals ; Mice ; Encephalitis Virus, Western Equine ; Ascites ; Immunosuppressive Agents ; Antibodies, Monoclonal ; Immunoglobulin M

People living with human immunodeficiency virus (PLHIV) are 100 times more at risk for cutaneous adverse drug reactions (ADRs). Acute generalized exanthematous pustulosis (AGEP) is a rare and severe cutaneous ADR associated with systemic involvement in 20% of cases.

This is a case of a 30-year-old male living with HIV admitted for acute gastroenteritis. Eight hours after initiation of intravenous ciprofloxacin and metronidazole, the onset of generalized monomorphic asymptomatic pustules was observed with associated weakness, fever, thrombocytosis, and neutrophilia. Ciprofloxacin was shifted to piperacillin-tazobactam. The patient was managed with intravenous hydrocortisone and oral cetirizine. Thereafter, the lesions remained stable in size and no new lesions occurred. The patient was referred to the dermatology service for further evaluation and management. A diagnostic workup was done which revealed subcorneal pustular dermatitis on histopathology, no fungal elements on periodic acid-Schiff stain, negative Gram stain, and no growth on culture. This case was diagnosed as AGEP secondary to ciprofloxacin. Dermatologic management consisted of oral antihistamines and topical steroids. The patient experienced generalized desquamation and gradual resolution of pustules over a two-week period with the eventual appearance of normal skin.

Ciprofloxacin is commonly used to treat opportunistic infections in the setting of HIV but it has never been documented to cause AGEP in such settings. Decreased CD4+ T-cell count (460 cells/µL) are factors associated with drug eruptions. Despite its toxic presentation, AGEP has a good prognosis with prompt withdrawal of the offending drug and supportive management.

Humans ; Herpesvirus 4, Human ; Epstein-Barr Virus Infections ; Carcinoma, Small Cell ; Nasopharynx/pathology* ; Carcinoma, Neuroendocrine/pathology* ; Nasopharyngeal Neoplasms/pathology*

Background and Objectives: Municipal solid waste workers (MSWWs) are important in the city’s waste management. With these vital contributions, they face unique occupational hazards and health risks. This study aims to determine the prevalence of occupational infections, such as soil-transmitted helminth infections (STHI) and hepatitis A virus (HAV), as well as the occurrence of Helicobacter pylori infection among the MSWWs of Baguio City. Methods: This cross-sectional analytic study collected data from volunteer MSWWs using a questionnaire to gather information on age, duration of employment, use of gloves in the workplace, and hand hygiene practices. Stool samples were obtained from participants and were analyzed for STHI using the Formalin Ether Concentration Technique (FECT). H. pylori infection was detected using the SD Bioline rapid antigen test kit on stool samples while blood samples were collected and tested for HAV antibodies using the Aria IgG/IgM rapid test kit. Results: Of the 44 volunteer MSWWs tested, 25 were infected with hazardous pathogens. Specifically, six workers (13.6%) were infected with STHI, four (9.1%) were infected with HAV and 15 (34.1%) were infected with H. pylori. Among those infected with STHI, Ascaris lumbricoides and Endolimax nana were the predominant species, each with a prevalence rate of 33.3%. In contrast, Blastocystis hominis and hookworm infections each had a prevalence rate of 16.7%. A significant association was found between STHI prevalence and the preference for alcohol hand rubs over hand washing, with a p-value of 0.008. Conclusion The analysis revealed a significant associat ion between the prevalence of STHI and the preference for alcohol hand rubs over hand washing, suggesting that MSWWs may have a false sense of security regarding their hygiene practices. The findings revealed the critical importance of proper hand washing in preventing STHI. Future research should expand data collection to encompass a broader range of socio-demographic, environmental, and lifestyle factors that may influence infection rates. Additionally, including a control group of individuals not exposed to waste management could help differentiate between factors specific to waste handling and those related to other occupations. This study emphasizes the need for collaborative efforts among researchers, public health authorities, and waste management agencies to enhance the health and safety of MSWWs while addressing broader public health concerns related to waste management practices.
Human ; Hepatitis A virus (HAV) ; Helicobacter pylori

The outbreak of monkeypox has become a global matter of concern since last year. Monkeypox is a zoonotic disease caused by monkeypox virus（MPXV）infection，and in addition to the typical symptom of rash，MPXV infection can cause a series of neurological manifestations，with the potential mechanisms of immune-mediated neurological damage after infection and direct invasion of the virus into the nervous system. This article reviews the neurological manifestations of MPXV infection，so as to facilitate the early identification and diagnosis of the neurological complications of MPXV infection and adopt appropriate prevention and treatment measures in a timely manner.
Monkeypox virus

The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.
Animals ; Chlorocebus aethiops ; Humans ; Interleukin-6/genetics* ; Janus Kinase 2/pharmacology* ; Infectious bronchitis virus/metabolism* ; STAT3 Transcription Factor/metabolism* ; Angiotensin-Converting Enzyme 2/pharmacology* ; Cytokine Receptor gp130/metabolism* ; Vero Cells ; Signal Transduction ; Inflammation ; RNA, Messenger

Objective To study the pathological types,expression of mismatch repair protein,human epidermal growth factor receptor 2(HER2),and Pan-TRK,and Epstein-Barr virus(EBV)infection in patients with colorectal cancer resected in Tibet. Methods A total of 79 patients with colorectal cancer resected in Tibet Autonomous Region People's Hospital from December 2013 to July 2021 were enrolled in this study.The clinical and pathological data of the patients were collected.The expression of mismatch repair protein,HER2,and Pan-TRK was detected by immunohistochemical(IHC)staining,and detection of HER2 gene by fluorescence in situ hybridization(FISH)in the patients with HER2 IHC results of 2+ or above.EBV was detected by in situ hybridization with EBV-encoded small RNA. Results A total of 79 colorectal cancer patients were included in this study,with the male-to-female ratio of 1.26:1 and the mean age of(57.06±12.74)years(24-83 years).Among them,4 patients received preoperative neoadjuvant therapy.Colonic cancer and rectal cancer occurred in 57(57/79,72.15%,including 31 and 26 in the right colon and left colon,respectively)and 22(22/79,27.85%)patients,respectively.The maximum diameter of tumor varied within the range of 1-20 cm,with the mean of(6.61±3.33)cm.Among the 79 colorectal cancer patients,75(75/79,94.94%)patients showed adenocarcinoma.Lymph node metastasis occurred in 12(12/21,57.14%)out of the 21 patients with severe tumor budding,13(13/23,56.52%)out of the 23 patients with moderate tumor budding,and 2(2/31,6.45%)out of the 31 patients with mild tumor budding,respectively.The lymph node metastasis rate showed differences between the patients with severe/moderate tumor budding and the patients with mild tumor budding(all P<0.001).The IHC staining showed that mismatch repair protein was negative in 10(10/65,15.38%)patients,including 5 patients with both MSH2 and MSH6 negative,4 patients with both MLH1 and PMS2 negative,and 1 patient with MSH6 negative.Pan-TRK was negative in 65 patients.The IHC results of HER2 showed 0 or 1+ in 60 patients and 2+ in 5 patients.FISH showed no positive signal in the 5 patients with HER2 IHC results of 2+.The detection with EBV-encoded small RNA showed positive result in 1(1/65,1.54%)patient. Conclusions Non-specific adenocarcinoma of the right colon is the most common in the patients with colorectal cancer resected in Tibet,and 15% of the patients showed mismatch repair protein defects.EBV-associated colorectal carcer is rare,Pan-TRK expression and HER2 gene amplification are seldom.The colorectal cancer patients with moderate and severe tumor budding are more likely to have lymph node metastasis.
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Adenocarcinoma ; Biomarkers, Tumor/genetics* ; Colorectal Neoplasms/pathology* ; DNA Mismatch Repair ; DNA-Binding Proteins/genetics* ; Epstein-Barr Virus Infections/diagnosis* ; Herpesvirus 4, Human/metabolism* ; In Situ Hybridization, Fluorescence ; Lymphatic Metastasis ; Tibet ; Young Adult ; Aged, 80 and over

Xiao Chaihu Decoction combined with Maxing Shigan Decoction is a classic herbal formula. All of them are derived from Treatise on Cold Damage(Shang Han Lun) by ZHANG Zhong-jing. This combination has the effects of harmonizing lesser yang, relieving exterior syndrome, clearing lung heat, and relieving panting. It is mainly used for treating the disease involving the triple-Yang combination of diseases and accumulation of pathogenic heat in the lung. Xiao Chaihu Decoction combined with Maxing Shigan Decoction is a classic combination for the treatment of exogenous diseases involving the triple-Yang combination. They are commonly used in exogenous diseases, especially in the north of China. This combination is also the main treatment strategy for coronavirus disease 2019(COVID-19) accompanied by fever and cough. Maxing Shigan Decoction is a classical herbal formula for treating the syndrome of phlegm-heat obstructing the lung. "Dyspnea after sweating" suggests the accumulation of pathogenic heat in the lung. Patients with mild symptoms may develop cough and asthma along with forehead sweating, and those in critical severe may develop whole-body sweating, especially the front chest. Modern medicine believes that the above situation is related to lung infection. "Mild fever" refers to syndromes rather than pathogenesis. It does not mean that the heat syndrome is not heavy, instead, it suggests that severe heat and inflammation have occurred. The indications of Xiao Chaihu Decoction combined with Maxing Shigan Decoction are as follows.(1) In terms of diseases, it is suitable for the treatment of viral pneumonia, bronchopneumonia, lobar pneumonia, mycoplasma pneumonia, COVID-19 infection, measles with pneumonia, severe acute respiratory syndrome(SARS), avian influenza, H1N1 influenza, chronic obstructive pulmonary disease with acute exacerbation, pertussis, and other influenza and pneumonia.(2) In terms of syndromes, it can be used for the syndromes of bitter mouth, dry pharynx, vertigo, loss of appetite, vexation, vomiting, and fullness and discomfort in the chest and hypochondrium. It can also be used to treat alternate attacks of chill and fever and different degrees of fever, as well as chest tightness, cough, asthma, expectoration, dry mouth, wanting cold drinks, feeling agitated, sweating, yellow urine, dry stool, red tongue, yellow or white fur, and floating, smooth, and powerful pulse, especially the right wrist pulse.
Animals ; Humans ; Cough ; Syndrome ; Influenza A Virus, H1N1 Subtype ; Influenza, Human ; COVID-19 ; Drugs, Chinese Herbal/pharmacology* ; Lung ; Pulmonary Disease, Chronic Obstructive/drug therapy* ; Asthma ; Critical Care ; Medicine, Chinese Traditional