Identification of subgenomic mRNAs derived from the coronavirus 1a/1b protein gene: Implications for coronavirus transcription.

Synthesis of coronavirus subgenomic mRNA (sgmRNA) is guided by the transcription regulatory sequence (TRS). sgmRNA derived from the body TRS (TRS-B) located at the 1a/1b protein gene is designated 1ab/sgmRNA. In the current study, we comprehensively identified the 1ab/sgmRNAs synthesized from TRS-Bs located at the 1a/1b protein genes of different coronavirus genera both in vitro and in vivo by RT‒PCR and sequencing. The results suggested that the degree of sequence homology between the leader TRS (TRS-L) and TRS-B may not be a decisive factor for 1ab/sgmRNA synthesis. This observation led us to revisit the coronavirus transcription mechanism and to propose that the disassociation of coronavirus polymerase from the viral genome may be a prerequisite for sgmRNA synthesis. Once the polymerase can disassociate at TRS-B, the sequence homology between TRS-L and TRS-B is important for sgmRNA synthesis. The study therefore extends our understanding of transcription mechanisms.

Identification of the protein coding capability of coronavirus defective viral genomes by mass spectrometry.

During coronavirus infection, in addition to the well-known coronavirus genomes and subgenomic mRNAs, an abundance of defective viral genomes (DVGs) can also be synthesized. In this study, we aimed to examine whether DVGs can encode proteins in infected cells. Nanopore direct RNA sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were employed. With the protein databases generated by nanopore direct RNA sequencing and the cell lysates derived from the RNA-protein pull-down assay, six DVG-encoded proteins were identified by LC-MS/MS based on the featured fusion peptides caused by recombination during DVG synthesis. The results suggest that the coronavirus DVGs have the capability to encode proteins. Consequently, future studies determining the biological function of DVG-encoded proteins may contribute to the understanding of their roles in coronavirus pathogenesis and the development of antiviral strategies.

Albuminuria Is Affected by Urinary Tract Infection: A Comparison between Biochemical Quantitative Method and Automatic Urine Chemistry Analyzer UC-3500.

The automated urine reagent strip test is a cost-effective tool for detecting albuminuria in patients. However, prior research has not investigated how urinary tract infections (UTIs) affect the test's accuracy. Therefore, this study aims to assess the impact of UTIs on albuminuria diagnosis using both the biochemical quantitative method and the test strip method of the Fully Automatic Urine Chemistry Analyzer, UC-3500 (Sysmex, Kobe, Japan). From March to December 2019, we prospectively collected midstream urine from adult female UTI patients before and after one week of cephalexin treatment. The urine samples were subjected to culture, routine urinalysis, and albuminuria diagnosis using the biochemical quantitative method and UC-3500. Albuminuria was defined as a urine albumin to creatinine ratio (UACR) ≥ 30 mg/g in the biochemical quantitative method. The results were compared between the two methods. Among fifty-four female patients (average age: 50.5 ± 4.4 years) with UTIs, 24 (44.44%) had transient albuminuria. The quantitative UACR significantly decreased after one week of antibiotic treatment (median: 53 mg/g to 9 mg/g; median difference: -0.54, p < 0.0001). UC-3500 exhibited a higher false positive rate for diagnosing albuminuria during UTIs (42%) compared to after treatment (19%). Its agreement with the biochemical quantitative method was moderate during UTI (κ = 0.49, 95% confidence interval [CI]: 0.24-0.73) and good after treatment (κ = 0.65, 95% CI: 0.45-0.86). UC-3500's accuracy in diagnosing albuminuria is influenced by UTIs, leading to either transient albuminuria or a false positive reaction of the test strip. UTI should be excluded or treated before its application in albuminuria screening.

Targeting the conserved coronavirus octamer motif GGAAGAGC is a strategy for the development of coronavirus vaccine.

BACKGROUND: Coronaviruses are pathogens of humans and animals that cause widespread and costly diseases. The development of effective strategies to combat the threat of coronaviruses is therefore a top priority. The conserved coronavirus octamer motif 5'GGAAGAGC3' exists in the 3' untranslated region of all identified coronaviruses. In the current study, we aimed to examine whether targeting the coronavirus octamer motif GGAAGAGC is a promising approach to develop coronavirus vaccine. METHODS: Plaque assays were used to determine the titers of mouse hepatitis virus (MHV)-A59 octamer mutant (MHVoctm) and wild-type (wt) MHV-A59 (MHVwt). Western blotting was used for the determination of translation efficiency of MHVoctm and MHVwt. Plaque assays and RT-qPCR were employed to examine whether MHVoctm was more sensitive to interferon treatment than MHVwt. Weight loss, clinical signs, survival rate, viral RNA detection and histopathological examination were used to evaluate whether MHVoctm was a vaccine candidate against MHVwt infection in BALB/c mice. RESULTS: In this study, we showed that (i) the MHVoctm with mutation of coronavirus octamer was able to grow to high titers but attenuated in mice, (ii) with the reduced multiplicity of infection (MOI), the difference in gene expression between MHVoctm and MHVwt became more evident in cultured cells, (iii) MHVoctm was more sensitive to interferon treatment than MHVwt and (iv) mice inoculated with MHVoctm were protected from MHVwt infection. CONCLUSIONS: Based on the results obtained from cultured cells, it was suggested that the synergistic effects of octamer mutation, multiplicity of infection and immune response may be a mechanism explaining the distinct phenotypes of octamer-mutated coronavirus in cell culture and mice. In addition, targeting the conserved coronavirus octamer motif is a strategy for development of coronavirus vaccine. Since the conserved octamer exists in all coronaviruses, this strategy of targeting the conserved octamer motif can also be applied to other human and animal coronaviruses for the development of coronavirus vaccines, especially the emergence of novel coronaviruses such as SARS-CoV-2, saving time and cost for vaccine development and disease control.

Evolution of the coronavirus spike protein in the full-length genome and defective viral genome under diverse selection pressures.

How coronaviruses evolve by altering the structures of their full-length genome and defective viral genome (DVG) under dynamic selection pressures has not been studied. In this study, we aimed to experimentally identify the dynamic evolutionary patterns of the S protein sequence in the full-length genome and DVG under diverse selection pressures, including persistence, innate immunity and antiviral drugs. The evolutionary features of the S protein sequence in the full-length genome and in the DVG under diverse selection pressures are as follows: (i) the number of nucleotide (nt) mutations does not necessarily increase with the number of selection pressures; (ii) certain types of selection pressure(s) can lead to specific nt mutations; (iii) the mutated nt sequence can be reverted to the wild-type nt sequence under the certain type of selection pressure(s); (iv) the DVG can also undergo mutations and evolve independently of the full-length genome; and (v) DVG species are regulated during evolution under diverse selection pressures. The various evolutionary patterns of the S protein sequence in the full-length genome and DVG identified in this study may contribute to coronaviral fitness under diverse selection pressures.

Biological characterization of coronavirus noncanonical transcripts in vitro and in vivo.

BACKGROUND: In addition to the well-known coronavirus genomes and subgenomic mRNAs, the existence of other coronavirus RNA species, which are collectively referred to as noncanonical transcripts, has been suggested; however, their biological characteristics have not yet been experimentally validated in vitro and in vivo. METHODS: To comprehensively determine the amounts, species and structures of noncanonical transcripts for bovine coronavirus in HRT-18 cells and mouse hepatitis virus A59, a mouse coronavirus, in mouse L cells and mice, nanopore direct RNA sequencing was employed. To experimentally validate the synthesis of noncanonical transcripts under regular infection, Northern blotting was performed. Both Northern blotting and nanopore direct RNA sequencing were also applied to examine the reproducibility of noncanonical transcripts. In addition, Northern blotting was also employed to determine the regulatory features of noncanonical transcripts under different infection conditions, including different cells, multiplicities of infection (MOIs) and coronavirus strains. RESULTS: In the current study, we (i) experimentally determined that coronavirus noncanonical transcripts were abundantly synthesized, (ii) classified the noncanonical transcripts into seven populations based on their structures and potential synthesis mechanisms, (iii) showed that the species and amounts of the noncanonical transcripts were reproducible during regular infection but regulated in altered infection environments, (iv) revealed that coronaviruses may employ various mechanisms to synthesize noncanonical transcripts, and (v) found that the biological characteristics of coronavirus noncanonical transcripts were similar between in vitro and in vivo conditions. CONCLUSIONS: The biological characteristics of noncanonical coronavirus transcripts were experimentally validated for the first time. The identified features of noncanonical transcripts in terms of abundance, reproducibility and variety extend the current model for coronavirus gene expression. The capability of coronaviruses to regulate the species and amounts of noncanonical transcripts may contribute to the pathogenesis of coronaviruses during infection, posing potential challenges in disease control. Thus, the biology of noncanonical transcripts both in vitro and in vivo revealed here can provide a database for biological research, contributing to the development of antiviral strategies.

Unveiling the biology of defective viral genomes in vitro and in vivo: implications for gene expression and pathogenesis of coronavirus.

BACKGROUND: Defective viral genome (DVG) is a truncated version of the full-length virus genome identified in most RNA viruses during infection. The synthesis of DVGs in coronavirus has been suggested; however, the fundamental characteristics of coronavirus DVGs in gene expression and pathogenesis have not been systematically analyzed. METHODS: Nanopore direct RNA sequencing was used to investigate the characteristics of coronavirus DVGs in gene expression including reproducibility, abundance, species and genome structures for bovine coronavirus in cells, and for mouse hepatitis virus (MHV)-A59 (a mouse coronavirus) in cells and in mice. The MHV-A59 full-length genomic cDNAs (~ 31 kilobases) were in vitro constructed to experimentally validate the origin of coronavirus DVG. The synthesis of DVGs was also experimentally identified by RT-PCR followed by sequencing. In addition, the alterations of DVGs in amounts and species under different infection environments and selection pressures including the treatment of antiviral remdesivir and interferon were evaluated based on the banding patterns by RT-PCR. RESULTS: The results are as follows: (i) the structures of DVGs are with diversity, (ii) DVGs are overall synthesized with moderate (MHV-A59 in cells) to high (BCoV in cells and MHV-A59 in mice) reproducibility under regular infection with the same virus inoculum, (iii) DVGs can be synthesized from the full-length coronavirus genome, (iv) the sequences flanking the recombination point of DVGs are AU-rich and thus may contribute to the recombination events during gene expression, (v) the species and amounts of DVG are altered under different infection environments, and (vi) the biological nature of DVGs between in vitro and in vivo is similar. CONCLUSIONS: The identified biological characteristics of coronavirus DVGs in terms of abundance, reproducibility, and variety extend the current model for coronavirus gene expression. In addition, the biological features of alterations in amounts and species of coronavirus DVGs under different infection environments may assist the coronavirus to adapt to the altered environments for virus fitness and may contribute to the coronavirus pathogenesis. Consequently, the unveiled biological features may assist the community to study the gene expression mechanisms of DVGs and their roles in pathogenesis, contributing to the development of antiviral strategy and public health.

An important call: Suggestion of using IL-10 as therapeutic agent for COVID-19 with ARDS and other complications.

The global coronavirus disease 2019 (COVID-19) pandemic has a detrimental impact on public health. COVID-19 usually manifests as pneumonia, which can progress into acute respiratory distress syndrome (ARDS) related to uncontrolled TH17 immune reaction. Currently, there is no effective therapeutic agent to manage COVID-19 with complications. The currently available anti-viral drug remdesivir has an effectiveness of 30% in SARS-CoV-2-induced severe complications. Thus, there is a need to identify effective agents to treat COVID-19 and the associated acute lung injury and other complications. The host immunological pathway against this virus typically involves the THαß immune response. THαß immunity is triggered by type 1 interferon and interleukin-27 (IL-27), and the main effector cells of the THαß immune response are IL10-CD4 T cells, CD8 T cells, NK cells, and IgG1-producing B cells. In particular, IL-10 exerts a potent immunomodulatory or anti-inflammatory effect and is an anti-fibrotic agent for pulmonary fibrosis. Concurrently, IL-10 can ameliorate acute lung injury or ARDS, especially those caused by viruses. Owing to its anti-viral activity and anti-pro-inflammatory effects, in this review, IL-10 is suggested as a possible treatment agent for COVID-19.

Risk of urinary tract infection symptoms recurrence in women: A prospective observational study.

OBJECTIVES: Urinary tract infection (UTI) symptoms recurrence is common with estimated rate of 30%-50% within 1 year. The study aimed to evaluate the potential risk factors for symptoms UTI recurrence in women at outpatient clinic in a prospective fashion. MATERIALS AND METHODS: This study was conducted from July 1, 2016, to June 30, 2019. Women who visited urological clinics with symptoms suggestive of UTI were invited to fill the questionnaire including baseline characteristics and Urinary Tract Infection Symptom Assessment questionnaire. Mid-stream urine samples of the participants were collected for urine analysis and urine culture. Phone interviews were done at 12 months' postclinic visit to inquire if the participants have any episode of UTI symptoms recurrence during the period of time. RESULTS: Among the 188 eligible patients, 183 patients (age = 50.0 ± 15.3 years old) were included in the analysis. There were 44 (24%) participants had UTI symptoms recurrent episodes during the 12-month follow-up. Further multivariate analysis revealed that menopause (odds ratio [OR] = 4.89, 95% confidence interval [CI] = 1.63-14.68, P = 0.005), history of UTI-related symptoms within 1 year before the episode OR = 3.79, 95% CI = 1.29-11.15, P = 0.016) and Escherichia coli infection (OR = 4.81, 95% CI = 1.51-15.28, P = 0.008) were significant risk factors for UTI symptoms recurrence during the 12 months' follow-up. CONCLUSION: Menopause, history of UTI-related symptoms within 1 year before this episode of UTI and E. coli infection in 12 months were potential risk factors for UTIs symptoms recurrence in women.

Snail Mucus Enhances Chemosensitivity of Triple-negative Breast Cancer Via Activation of the Fas Pathway.

BACKGROUND/AIM: The poor prognosis and chemoresistance of patients with triple-negative breast cancer (TNBC) urge the development of new therapeutic strategies. Snail mucus has shown its ability against inflammation, a process closely related to tumorigenesis, suggesting a potential anti-cancer activity. MATERIALS AND METHODS: The effect and mechanisms of snail mucus on cell viability were determined by IncuCyte Live-cell analysis and molecular biological methods. The anti-cancer fractions of snail mucus were isolated and identified by medium pressure liquid chromatography (MPLC) and nuclear magnetic resonance (NMR) spectrometry analysis. RESULTS: Snail mucus significantly decreased the viability of TNBC cells with relatively lower cytotoxicity to normal breast epithelial cells and enhanced their response to chemotherapy through activation of Fas signaling by suppressing nucleolin. Two peptide fractions have been identified as the anti-cancer ingredients of the snail mucus. CONCLUSION: Snail mucus can induce programmed cell death via the extrinsic apoptotic pathway and has therapeutic potential by achieving a chemo-sensitizing effect in TNBCs.

Automated Urine Particle Analyzer UF-1000i Can Pre-Estimate the Treatment Response of Women's Uncomplicated Urinary Tract Infections to Antibiotics.

PURPOSE: To evaluate the ability of bacterial scatter diagrams generated from the automated urine particle analyzer (UF-1000i, Sysmex, Kobe, Japan) to pre-estimate the treatment efficacy of oral cefalexin in treating women with uncomplicated urinary tract infection (uUTI). MATERIALS AND METHODS: Over 3 years, women 20-80 years old with symptoms suggestive of uUTI (Urinary Tract Infection Symptoms Assessment symptom score, ,UTISA > 3) and bacteriuria (bacterial count ≥ 100/uL) were enrolled. After informed consent, patients took cephalexin 500mg 4 times/day for 7 days. The voided urine specimens were classified into rods or cocci/mixed group automatically through the built-in software of the UF1000i. Patients were followed up with UTISA on the 3rd day after treatment and returned to the clinic on the 7th day and followed for additional UTISA and urine analysis. Symptom and laboratory improvement were defined as UTISA < 4 and bacterial count < 100/uL, respectively, on the 7th day. RESULTS: Of 99 women (age: 49.91 ±15.32 years) eligible for analysis, 80 were classified as having urine that contained rods and 19 as cocci/mixed. Symptom improvement was observed in 62 women in the rods group and 11 women in the cocci/mixed group (p = 0.08). Laboratory improvement was noted in 64 women in the rods group and 10 women in the cocci/mixed group (p = 0.01). On day 7, treatment success with both symptom and laboratory improvement was more observed in rods than in cocci/mixed group (61.3% vs. 26.3%, p < 0.01). CONCLUSION: The automatic urine particle analyzer can pre-estimate the treatment response of antibiotics in women with uUTI.

A performance comparison of the fully automated urine particle analyzer UF-5000 with UF-1000i and Gram staining in predicting bacterial growth patterns in women with uncomplicated urinary tract infections.

BACKGROUND: The aim of this study was to compare the performance of the new flow cytometer UF-5000 with the UF-1000i and Gram staining for determining bacterial patterns in urine samples. METHODS: Women who attended our clinic with symptoms suggestive of urinary tract infection were enrolled in the study. Mid-stream urine samples were collected for gram staining, urine analysis and urine cultures. Bacterial patterns were classified using the UF-1000i (none, cocci bacteria or rods/mixed growth), the UF-5000 (none, cocci, rods or mixed growth) and Gram staining. RESULTS: Among the 102 included samples, there were 10 g-positive cocci, 2 g-positive bacilli, 66 g-negative rods, and 24 mixed growth. The sensitivity/specificity of the UF-1000i was 81.8/91.1% for gram-negative rods and 23.5/96.9% for cocci/mixed. The sensitivity/specificity of the UF-5000 was 80.0/88.2% for gram negative rods and 70.0/86.5% for gram-positive cocci. CONCLUSIONS: The UF-5000 demonstrated good sensitivity and specificity for Gram-negative bacilli and demonstrated an improved sensitivity for detecting Gram-positive cocci compared with the UF-1000i.

Down-regulating microRNA-20a regulates CDH1 to protect against cerebral ischemia/reperfusion injury in rats.

Studies have extensively focused on the involvement of microRNAs (miRNAs) in cerebral ischemia/reperfusion (I/R) injury but not much on the specific role of miR-20a. Hence, this study is purposed to decipher whether miR-20a could regulate cadherin 1 (CDH1) to affect cerebral I/R injury in rats. Rat transient middle cerebral artery occlusion model (MCAO) was established. Rats were injected with lentiviral solution containing miR-20a inhibitor, or overexpressed CDH1 or combined depleted miR-20a and CDH1 to explore their roles in cerebral I/R injury. Oxidative stress-related factors, miR-20a, CDH1, nuclear factor-kappaB (NF-κB) and Nestin expression in brain tissues were detected by RT-qPCR and western blot assay. The target relation between miR-20a and CDH1 was predicted by online website and further confirmed by luciferase activity assay. In rats with cerebral I/R injury, increased miR-20a and decreased CDH1 were found in brain tissues. Reduction of miR-20a or elevation of CDH1 attenuated behavior function in MCAO rats. Inhibiting miR-20a or restoring CDH1 restrained oxidative stress, attenuated pathological damage of neurons, promoted neuron survival, and down-regulated NF-κB and Nestin expression in brain tissues of MCAO rats. CDH1 was determined to a target gene of miR-20a. This study elucidates that down-regulating miR-20a elevates CDH1 to protect neurons from cerebral I/R injury, which paves a new way for treatment of cerebral I/R injury.

The association of vitamin D with hepatitis B virus replication: Bystander rather than offender.

BACKGROUND/PURPOSE: Low vitamin D is frequent in hepatitis B virus (HBV)-infected patients and several studies show an inverse association of serum vitamin D level with HBV viral load. However, the causal relationship remains unclear. METHODS: HBV carriers receiving regular 6-month surveillance without current antiviral treatment or cirrhosis were invited to participate into this trial. The markers of HBV replication included serum HBV DNA and quantitative HBsAg (qHBsAg) levels. Those with undetectable HBV DNA or sufficient vitamin D levels, cancer or electrolyte imbalance were excluded. The eligible subjects were randomized to receive either vitamin D supplement 2000 IU per day for 2 months (vitamin D group) or none (control group). RESULTS: A total of 196 HBV carriers (93 males and 103 females; mean age 51.9 ± 10.0 years) were screened. Of them, 28 patients had undetectable serum HBV DNA levels, which is defined as spontaneous viral clearance. The vitamin D levels were not different between patients with detectable HBV DNA and those without (p = 0.18). After exclusion, 149 patients were randomized to two groups: 75 in vitamin D group and 74 in control group. After 2 months vitamin D supplement, the serum vitamin D levels were significantly higher in the vitamin D group than the control group (p < 0.001). However, the serum qHBsAg and HBV DNA levels were comparable between these two groups. CONCLUSION: There is no causal relationship between vitamin D and HBV replication. The role of liver reserve on serum vitamin D levels in patients with chronic HBV infection needs further investigation.

Rapid differentiation of cocci/mixed bacteria from rods in voided urine culture of women with uncomplicated urinary tract infections.

OBJECTIVES: To evaluate the ability of laser flow cytometry to predict cocci/mixed growth in the pre-analytical phase of urine specimens. METHODS: We retrospectively reviewed urine samples from women with uncomplicated urinary tract infections from urologic clinics for study. Urine analyses were performed with laser flow cytometry (UF1000i, Sysmex, Kobe, Japan) and then diagrams were generated (forward scatter vs. fluorescent light scatter). Each specimen (bacteria count >357 BACT/µL) was classified as either cocci bacteria or rods/mixed growth according to the diagrams. Standard urine cultures were performed, and the agreement between cultures and the UF1000i interpretations was analyzed with kappa statistics. RESULTS: Finally, 491 specimens met the criteria for analysis. Among the 376 specimens with single bacteria growth, there were 26 gram-positive cocci (13 Streptococci spp., 7 Staphylococci spp., 6 Enterococci spp.), 1 gram-positive rods (Corynebacterium spp.), and 349 gram-negative rods (273 Escherichia coli, 33 Klebsiella spp., 29 Proteus spp., 6 Citrobacter spp., 4 Enterobacter spp., 3 Pseudomonas spp., and 1 Providencia spp.). There were 115 specimens with two bacteria species or more that were regarded as mixed growth. Agreement of rods or cocci/mixed growth between the laser flow cytometry and urine cultures yielded a kappa value of 0.58. The positive and negative predictive rate of the UF1000i for cocci/mixed growth in voided urine culture was 81.8% and 84.7%, respectively. CONCLUSIONS: Through laser flow cytometry, we can predict growth of cocci/mixed growth in the pre-analytical phase of urine culture, thus avoiding unnecessary urine culture and waiting time.

Growth-differentiation factor-15 and major cardiac events.

BACKGROUND: Growth-differentiation factor (GDF)-15 is a strong predictor of cardiovascular events in patients with ST-elevation myocardial infarction (STEMI). However, the effects of GDF-15 on left ventricular (LV) remodeling have not been clearly elucidated. The aim of this study is to investigate whether GDF-15 will be of benefit in predicting LV remodeling, heart failure and death in patients with STEMI. METHODS: The authors enrolled 216 patients with STEMI who received measurement of GDF-15 level on day 2 of hospitalization. Echocardiographic studies were performed at baseline and were repeated 6 months later. Clinical events, including all-cause death and readmission for heart failure, were followed up for a maximum of 3 years. RESULTS: Patients with GDF-15 levels above the median had lower LV ejection fraction at baseline (43.9% versus 48.0%, P = 0.041) and at 6 months (51.5% versus 56.9%, P = 0.025). In univariable regression model, log-transformed GDF-15 level was not a predictor of increase in LV end-diastolic volume index at 6 months (P = 0.767). Kaplan-Meier survival curves showed that the combination of high GDF-15 and high N-terminal pro-B-type natriuretic peptide was a strong predictor of death and heart failure (P < 0.001). In multivariable Cox regression model, the independent predictors of death and heart failure were age, GDF-15 level and diabetes mellitus. CONCLUSIONS: High GDF-15 level is a strong predictor of death and heart failure in patients with STEMI. Although patients with higher GDF-15 levels tend to have lower LV ejection fraction, they have similar degree of the increase in LV end-diastolic volume index at 6 months.