The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component ...have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.
Introduction
Pregnancy is a time of increased vulnerability to mental health disorders. Additionally, the COVID-19 pandemic has increased the incidence of depression and anxiety. Thus, we aimed to ...assess mental health and associated healthy behaviors of pregnant people in California during the pandemic in order to contextualize prenatal well-being during the first pandemic of the twenty-first century.
Methods
We conducted an online cross-sectional study of 433 pregnant people from June 6 through July 29, 2020. We explored 3 hypotheses: (1) mental health would be worse during the pandemic than in general pregnant samples to date; (2) first-time pregnant people would have worse mental health; and (3) healthy behaviors would be positively related to mental health.
Results
Many of our participants (22%) reported clinically significant depressive symptoms and 31% reported clinically significant anxiety symptoms. Multiparous pregnant people were more likely to express worries about their own health and wellbeing and the process of childbirth than were primiparous pregnant people. Additionally, as pregnancy advanced, sleep and nutrition worsened, while physical activity increased. Lastly, anxious-depressive symptomology was significantly predictive of participant sleep behaviors, nutrition, and physical activity during the past week.
Discussion
Pregnant people had worse mental health during the pandemic, and this was associated with worse health-promoting behaviors. Given that the COVID-19 pandemic and associated risks are likely to persist due to low vaccination rates and the emergence of variants with high infection rates, care that promotes mental and physical well-being for the pregnant population should be a public health priority.
Significance
Consistent with other studies, we found that anxiety, depression, and stress levels in our pregnant cohort were higher than reported pre-pandemic and that anxious-depressive symptomology predicted worse sleep, poorer nutrition, and less physical activity. New in this study was that these relationships were the same for primiparous and multiparous pregnant people, did not change with gestational age, and that while sleep and nutrition worsened throughout pregnancy, physical activity actually increased, regardless of anxious-depressive symptomology. Also new to this study was that multiparous pregnant people had more worries about their and their baby’s wellbeing than first time mothers.
Aims
Dose escalation at the initiation of allopurinol therapy can be protracted and resource intensive. Tools to predict the allopurinol doses required to achieve target serum urate concentrations ...would facilitate the implementation of more efficient dose‐escalation strategies. The aim of this research was to develop and externally evaluate allopurinol dosing tools, one for use when the pre‐urate‐lowering therapy serum urate is known (Easy‐Allo1) and one for when it is not known (Easy‐Allo2).
Methods
A revised population pharmacokinetic‐pharmacodynamic model was developed using data from 653 people with gout. Maintenance doses to achieve the serum urate target of <0.36 mmol L−1 in >80% of individuals were simulated and evaluated against external data. The predicted and observed allopurinol doses were compared using the mean prediction error (MPE) and root mean square error (RMSE). The proportion of Easy‐Allo predicted doses within 100 mg of the observed was quantified.
Results
Allopurinol doses were predicted by total body weight, baseline urate, ethnicity and creatinine clearance. Easy‐Allo1 produced unbiased and suitably precise dose predictions (MPE 2 mg day−1 95% confidence interval CI −13‐17, RMSE 91%, 90% within 100 mg of the observed dose). Easy‐Allo2 was positively biased by about 70 mg day−1 and slightly less precise (MPE 70 mg day−1 95% CI 52‐88, RMSE 131%, 71% within 100 mg of the observed dose).
Conclusions
The Easy‐Allo tools provide a guide to the allopurinol maintenance dose requirement to achieve the serum urate target of <0.36 mmol L−1 and will aid in the development of novel dose‐escalation strategies for allopurinol therapy.
Carbapenem-resistant Enterobacteriaceae (CRE) are increasingly reported worldwide as a cause of infections with high-mortality rates. Assessment of the US epidemiology of CRE is needed to inform ...national prevention efforts.
To determine the population-based CRE incidence and describe the characteristics and resistance mechanism associated with isolates from 7 US geographical areas.
Population- and laboratory-based active surveillance of CRE conducted among individuals living in 1 of 7 US metropolitan areas in Colorado, Georgia, Maryland, Minnesota, New Mexico, New York, and Oregon. Cases of CRE were defined as carbapenem-nonsusceptible (excluding ertapenem) and extended-spectrum cephalosporin-resistant Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae complex, Klebsiella pneumoniae, or Klebsiella oxytoca that were recovered from sterile-site or urine cultures during 2012-2013. Case records were reviewed and molecular typing for common carbapenemases was performed.
Demographics, comorbidities, health care exposures, and culture source and location.
Population-based CRE incidence, site-specific standardized incidence ratios (adjusted for age and race), and clinical and microbiological characteristics.
Among 599 CRE cases in 481 individuals, 520 (86.8%; 95% CI, 84.1%-89.5%) were isolated from urine and 68 (11.4%; 95% CI, 8.8%-13.9%) from blood. The median age was 66 years (95% CI, 62.1-65.4 years) and 284 (59.0%; 95% CI, 54.6%-63.5%) were female. The overall annual CRE incidence rate per 100<000 population was 2.93 (95% CI, 2.65-3.23). The CRE standardized incidence ratio was significantly higher than predicted for the sites in Georgia (1.65 95% CI, 1.20-2.25; P < .001), Maryland (1.44 95% CI, 1.06-1.96; P = .001), and New York (1.42 95% CI, 1.05-1.92; P = .048), and significantly lower than predicted for the sites in Colorado (0.53 95% CI, 0.39-0.71; P < .001), New Mexico (0.41 95% CI, 0.30-0.55; P = .01), and Oregon (0.28 95% CI, 0.21-0.38; P < .001). Most cases occurred in individuals with prior hospitalizations (399/531 75.1%; 95% CI, 71.4%-78.8%) or indwelling devices (382/525 72.8%; 95% CI, 68.9%-76.6%); 180 of 322 (55.9%; 95% CI, 50.0%-60.8%) admitted cases resulted in a discharge to a long-term care setting. Death occurred in 51 (9.0%; 95% CI, 6.6%-11.4%) cases, including in 25 of 91 cases (27.5%; 95% CI, 18.1%-36.8%) with CRE isolated from normally sterile sites. Of 188 isolates tested, 90 (47.9%; 95% CI, 40.6%-55.1%) produced a carbapenemase.
In this population- and laboratory-based active surveillance system in 7 states, the incidence of CRE was 2.93 per 100<000 population. Most CRE cases were isolated from a urine source, and were associated with high prevalence of prior hospitalizations or indwelling devices, and discharge to long-term care settings.
pv.
(Psa) and
pv.
(Pfm) are closely related pathovars infecting kiwifruit, but Psa is considered one of the most important global pathogens, whereas Pfm is not. In this study of
'Hayward' responses ...to the two pathovars, the objective was to test whether differences in plant defense responses mounted against the two pathovars correlated with the contrasting severity of the symptoms caused by them. Results showed that Psa infections were always more severe than Pfm infections, and were associated with highly localized, differential expression of phytohormones and putative defense gene transcripts in stem tissue closest to the inoculation site. Phytohormone concentrations of jasmonic acid (JA), jasmonate isoleucine (JA-Ile), salicylic acid (SA) and abscisic acid were always greater in stem tissue than in leaves, and leaf phytohormones were not affected by pathogen inoculation. Pfm inoculation induced a threefold increase in SA in stems relative to Psa inoculation, and a smaller 1.6-fold induction of JA. Transcript expression showed no effect of inoculation in leaves, but Pfm inoculation resulted in the greatest elevation of the SA marker genes, PR1 and glucan endo-1,3-beta-glucosidase (β-1,3-glucosidase) (32- and 25-fold increases, respectively) in stem tissue surrounding the inoculation site. Pfm inoculation also produced a stronger response than Psa inoculation in localized stem tissue for the SA marker gene PR6, jasmonoyl-isoleucine-12-hydrolase (JIH1), which acts as a negative marker of the JA pathway, and APETALA2/Ethylene response factor 2 transcription factor (AP2 ERF2), which is involved in JA/SA crosstalk. WRKY40 transcription factor (a SA marker) was induced equally in stems by wounding (mock inoculation) and pathovar inoculation. Taken together, these results suggest that the host appears to mount a stronger, localized, SA-based defense response to Pfm than Psa.
Our previous genome-wide association study (GWAS) for sagittal nonsyndromic craniosynostosis (sNCS) provided important insights into the genetics of midline CS. In this study, we performed a GWAS for ...a second midline NCS, metopic NCS (mNCS), using 215 non-Hispanic white case-parent triads. We identified six variants with genome-wide significance (
P
≤ 5 × 10
–8
): rs781716 (
P
= 4.71 × 10
–9
; odds ratio OR = 2.44) intronic to
SPRY3
; rs6127972 (
P
= 4.41 × 10
–8
; OR = 2.17) intronic to
BMP7;
rs62590971 (
P
= 6.22 × 10
–9
; OR = 0.34), located ~ 155 kb upstream from
TGIF2LX
; and rs2522623, rs2573826, and rs2754857, all intronic to
PCDH11X
(
P
= 1.76 × 10
–8
, OR = 0.45;
P
= 3.31 × 10
–8
, OR = 0.45;
P
= 1.09 × 10
–8
, OR = 0.44, respectively). We performed a replication study of these variants using an independent non-Hispanic white sample of 194 unrelated mNCS cases and 333 unaffected controls; only the association for rs6127972 (
P
= 0.004, OR = 1.45; meta-analysis
P
= 1.27 × 10
–8
, OR = 1.74) was replicated. Our meta-analysis examining single nucleotide polymorphisms common to both our mNCS and sNCS studies showed the strongest association for rs6127972 (
P
= 1.16 × 10
–6
). Our imputation analysis identified a linkage disequilibrium block encompassing rs6127972, which contained an enhancer overlapping a CTCF transcription factor binding site (chr20:55,798,821–55,798,917) that was significantly hypomethylated in mesenchymal stem cells derived from fused metopic compared to open sutures from the same probands. This study provides additional insights into genetic factors in midline CS.
Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) cause acute respiratory tract disease and play an important role in the initiation and exacerbation of asthma. Our recently ...published data demonstrate that loss of viral control in nasal epithelial cells (NECs) from pre-asthmatic children is viral specific and IFN-independent. However this has not yet been established in asthmatic adults. Virus-induced Endoplasmic Reticulum (ER) stress leads to the activation of the unfolded protein response (UPR), and can contribute to viral replication and pathogenesis. The objectives of this study are to identify if intrinsic innate immune deficiencies exist in asthmatic adult NECs in response to RSV and hMPV, and whether this is associated with the ER stress response.
NECs were collected from healthy (n=10) and asthmatic (n=10) adults and cultured as submerged monolayers. Cells were infected with RSV-A2 or hMPV-CAN97/83 strains at high (3) multiplicity of infection. IFN-β, ER stress markers, caspase 3/7 activity and viral replication were quantified 24h post-infection.
Increased viral replication of hMPV, but not RSV, was observed in asthmatic NECs compared to healthy controls, although the IFN-β response was similar. qPCR analysis revealed an elevated expression of ER chaperone, Grp78 (p<0.05), spliced X-box binding protein 1 (sXBP1) (p<0.01) and CHOP (p<0.05) in hMPV-infected asthmatic NECs compared to healthy NECs or asthmatic NECs infected with RSV. Caspase 3/7 activation by hMPV indicated a 2-fold increase in apoptosis of asthmatic NECs compared to healthy NECs.
Our data indicate that asthmatic NECs are more susceptible to infection by hMPV, but not RSV, despite a normal IFN-β response. Elevated ER stress and apoptosis were associated with elevated hMPV replication in asthmatic NECs. The stress response of NECs was virus-specific and associated with disease status, thus highlighting the importance of the UPR in viral infections in asthmatic individuals.
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