Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering.

The COVID-19 pandemic has had a significant impact on human health management. A rapid diagnosis of SARS-CoV2 at the point-of-care (POC) is critical to prevent disease spread. As a POC device for remote settings, a LFIA should not require cold-chain maintenance and should be kept at normal temperatures. Antigen stability can be enhanced by addressing instability issues when dealing with fragile components, such as proteinaceous capture antigens. This study used immunologically guided protein engineering to enhance the capture nucleocapsid (NP) antigen stability of SARS-CoV2. A search of the IEDB database revealed that antibodies detecting epitopes are almost uniformly distributed over NP1-419. In contrast, N-terminal stretches of NP1-419 are theoretically more unstable than C-terminal stretches. We identified NP250-365 as a NP stretch with a low instability index and B-cell epitopes. Apart from NP1-419, two other variants (NP121-419 and NP250-365) were cloned, expressed, and purified. The degradation pattern of the proteins was observed on SDS-PAGE after three days of stability studies at -20 °C, 4 °C, and 37 °C. NP1-419 was the most degraded while NP250-365 exhibited the least degradation. Also, NP1-419, NP250-365, and NP121-419 reacted with purified antibodies from COVID-19 patient serum. Our results suggest that NP250-365 may be used as a stable capture antigen in LFIA devices to detect COVID-19.

Impact of COVID-19 pandemic early response measures on myocardial infarctions and acute cardiac care in Singapore.

The COVID -19 pandemic impacted acute myocardial infarction (AMI) attendances, ST-elevation myocardial infarction (STEMI) treatments, and outcomes. We collated data from majority of primary percutaneous coronary intervention (PPCI)-capable public healthcare centres in Singapore to understand the initial impact COVID-19 had on essential time-critical emergency services. We present data comparisons from 'Before Disease Outbreak Response System Condition (DORSCON) Orange', 'DORSCON Orange to start of circuit breaker (CB)', and during the first month of 'CB'. We collected aggregate numbers of weekly elective PCI from four centres and AMI admissions, PPCI, and in-hospital mortality from five centres. Exact door-to-balloon (DTB) times were recorded for one centre; another two reported proportions of DTB times exceeding targets. Median weekly elective PCI cases significantly decreased from 'Before DORSCON Orange' to 'DORSCON Orange to start of CB' (34 vs 22.5, P = 0.013). Median weekly STEMI admissions and PPCI did not change significantly. In contrast, the median weekly non-STEMI (NSTEMI) admissions decreased significantly from 'Before DORSCON Orange' to 'DORSCON Orange to start of CB' (59 vs 48, P = 0.005) and were sustained during CB (39 cases). Exact DTB times reported by one centre showed no significant change in the median. Out of three centres, two reported significant increases in the proportion that exceeded DTB targets. In-hospital mortality rates remained static. In Singapore, STEMI and PPCI rates remained stable, while NSTEMI rates decreased during DORSCON Orange and CB. The severe acute respiratory syndrome (SARS) experience may have helped prepare us to maintain essential services such as PPCI during periods of acute healthcare resource strain. However, data must be monitored and increased pandemic preparedness measures must be explored to ensure that AMI care is not adversely affected by continued COVID fluctuations and future pandemics.

One-year outcomes of patients with ST-segment elevation myocardial infarction during the COVID-19 pandemic.

The pandemic has led to adverse short-term outcomes for patients with ST-segment elevation myocardial infarction (STEMI). It is unknown if this translates to poorer long-term outcomes. In Singapore, the escalation of the outbreak response on February 7, 2020 demanded adaptation of STEMI care to stringent infection control measures. A total of 321 patients presenting with STEMI and undergoing primary percutaneous coronary intervention at a tertiary hospital were enrolled and followed up over 1-year. They were allocated into three groups based on admission date-(1) Before outbreak response (BOR): December 1, 2019-February 6, 2020, (2) During outbreak response (DOR): February 7-March 31, 2020, and (3) control group: November 1-December 31, 2018. The incidence of cardiac-related mortality, cardiac-related readmissions, and recurrent coronary events were examined. Although in-hospital outcomes were worse in BOR and DOR groups compared to the control group, there were no differences in the 1-year cardiac-related mortality (BOR 8.7%, DOR 7.1%, control 4.8%, p = 0.563), cardiac-related readmissions (BOR 15.1%, DOR 11.6%, control 12.0%, p = 0.693), and recurrent coronary events (BOR 3.2%, DOR 1.8%, control 1.2%, p = 0.596). There were higher rates of additional PCI during the index admission in DOR, compared to BOR and control groups (p = 0.027). While patients admitted for STEMI during the pandemic may have poorer in-hospital outcomes, their long-term outcomes remain comparable to the pre-pandemic era.

Impact of the COVID-19 Pandemic on Door-to-Balloon Time for Primary Percutaneous Coronary Intervention　- Results From the Singapore Western STEMI Network.

BACKGROUND: Little is known about the effect of the coronavirus disease 2019 (COVID-19) pandemic and the outbreak response measures on door-to-balloon time (D2B). This study examined both D2B and clinical outcomes of patients with STEMI undergoing primary percutaneous coronary intervention (PPCI).Methods and Results:This was a retrospective study of 303 STEMI patients who presented directly or were transferred to a tertiary hospital in Singapore for PPCI from October 2019 to March 2020. We compared the clinical outcomes of patients admitted before (BOR) and during (DOR) the COVID-19 outbreak response. The study outcomes were in-hospital death, D2B, cardiogenic shock and 30-day readmission. For direct presentations, fewer patients in the DOR group achieved D2B time <90 min compared with the BOR group (71.4% vs. 80.9%, P=0.042). This was more apparent after exclusion of non-system delay cases (DOR 81.6% vs. BOR 95.9%, P=0.006). Prevalence of both out-of-hospital cardiac arrest (9.5% vs. 1.9%, P=0.003) and acute mitral regurgitation (31.6% vs. 17.5%, P=0.006) was higher in the DOR group. Mortality was similar between groups. Multivariable regression showed that longer D2B time was an independent predictor of death (odds ratio 1.005, 95% confidence interval 1.000-1.011, P=0.029). CONCLUSIONS: The COVID-19 pandemic and the outbreak response have had an adverse effect on PPCI service efficiency. The study reinforces the need to focus efforts on shortening D2B time, while maintaining infection control measures.

Isolated right ventricle infarction.

We described two patients who were successfully resuscitated from out-of-hospital cardiac arrest. Their ECGs showed ST elevations in V1 and aVR, as well as diffuse ST depression. Their ST elevation in V1 was noted to be greater than in aVR. While one patient was found to have an occlusion of the right ventricular (RV) branch of the right coronary artery, the other was found to have an occlusion of a proximal non-dominant right coronary artery supplying the RV branch. Successful primary percutaneous coronary intervention was performed for each patient with angioplasty and implantation of a drug-eluting stent. Both patients made good physical and neurological recovery.

Acupuncture ( zhen jiǔ) - an emerging adjunct in routine oral care.

Acupuncture ( Zhen Jiǔ) ('acus' (needle) + 'punctura' (to puncture)) is the stimulation of specific points along the skin of the body involving various methods such as penetration by thin needles or the application of heat, pressure, or laser light. Acupuncture ( Zhen Jiǔ) aims to treat a range of medical and dental ailments, though is most commonly used for pain relief. This article reviews about the various possible roles of acupuncture ( Zhen Jiǔ) in clinical dental practice. Acupuncture ( Zhen Jiǔ) has potential in supplementing conventional treatment procedures by its diverse applicability outreach. Role of acupuncture ( Zhen Jiǔ) in dental practice has been well supported by clinical trials. Its role in alleviating facial pain, pre-operative and post-operative dental pain has led to its widespread application. Its role as sole analgesic for treatment procedure has to be tested. It's It is a thought that acupuncture ( Zhen Jiǔ) may prove an indispensible supplement to conventional treatment modalities and more of clinical trials and studies are required to prove the efficacy. Acupuncture ( Zhen Jiǔ) is not a miracle cure and is not going to replace the drill. However, the technique can be a supplement to conventional treatments in TMDs, facial pain, pain management Sjoegrens syndrome, and in phobias and anxiety. The application and use of Acupuncture ( Zhen Jiǔ) comes with some side effects. Proper training needs to be obtained before commencement of any procedure related to acupuncture ( Zhen Jiǔ). Various training programs are offered to train clinical practitioners the apt method to use acupuncture ( Zhen Jiǔ).

Comparative Study of Blood Loss, Quality of Surgical Field and Duration of Surgery in Maxillofacial Cases with and without Hypotensive Anesthesia.

BACKGROUND: Oral and maxillofacial surgeons are concern about the intraoperative bleeding because blood supply to face is very generous. Increased blood loss may lead to the need for transfusion of blood and blood products and hence patients are exposed to the risks associated with it. Hence, this study was undertaken to compare whether there is any significant difference in blood loss, surgical field and duration of surgery in patients undergoing maxillofacial surgeries with and without hypotensive anesthesia. MATERIALS AND METHODS: The study was conducted on patients undergoing various maxillofacial surgeries. The patients were randomly divided into two groups, the study group and the control group. In the study group patients, induced hypotension was used in order to maintain systolic pressure of 80-90 mm Hg. In the control group patients, normotensive anesthesia was used. Estimation of blood loss (EBS), quality of the surgical field and duration of surgery was calculated for both groups in three types of surgical procedures. Statistical analysis was performed by using two-tailed Pearson correlation test. RESULTS: EBS was found to be significantly less in all the surgical procedures carried out under hypotensive anesthesia (P < 0.05). The quality of the surgical field was better in cases with induced hypotension, but there was no significant difference in duration of the procedures with and without induced hypotension. CONCLUSION: Our study shows that hypotensive anesthesia can be safely used in almost all maxillofacial surgical procedures with reduced blood loss and improved surgical field.

Heart protection by combination therapy with esmolol and milrinone at late-ischemia and early reperfusion.

INTRODUCTION: The present study determined whether late-ischemia/early reperfusion therapy with the ß(1)-adrenergic receptor (AR) blocker esmolol and phosphodiesterase III inhibitor milrinone reduced left ventricular (LV) myocardial infarct size (IS). METHODS AND RESULTS: In an ischemia/reperfusion rat model (30-min ischemia/4-hr reperfusion), esmolol, milrinone or esmolol + milrinone were intravenous (IV) infused over 10 min (from the last 5 min of ischemia to the first 5 min of reperfusion). LV-IS were 48.9 ± 8.9%, 41.5 ± 5.4%, 25.8 ± 7.7% and 16.8 ± 7.3% for saline, esmolol, milrinone, and esmolol + milrinone, respectively (n = 12/group). Esmolol + milrinone further reduced LV-IS compared with esmolol or milrinone alone (p < 0.05). LV-IS-reduction induced by esmolol + milrinone was eliminated in the presence of protein kinase A-(PKA)-inhibitor (Rp-cAMPS) or Akt-inhibitor (AKT 1/2 kinase inhibitor). In mixed rat ventricular cardiomyocyte cultures, intra-ischemic application of esmolol, milrinone or esmolol + milrinone reduced myocyte death rates by 5.5%, 13.3%, and 16.8%, respectively, compared with saline (p < 0.01). This cell protective effect by esmolol + milrinone was abrogated in the presence of PKA-inhibitor or Akt-inhibitor. Esmolol, milrinone or esmolol + milrinone increased myocardial PKA activity by 22%, 28% and 59%, respectively, compared with saline (n = 6, p < 0.01). No non-specific adverse effect of Rp-cAMPS on myocytes was identified in a purified myocyte preparation during hypoxia/re-oxygenation. Antiapoptotic pathways were assessed by measuring myocardial phosphorylated Akt (pAkt) levels combined with terminal dUTP nick-end labelling staining analysis. Ten minutes following infusion of esmolol, milrinone or esmolol + milrinone, there were 1.7-, 2.7-, and 6-fold increase in tissue pAkt levels, respectively. This esmolol + milrinone induced pAkt activation was abolished in the presence of PKA inhibitor. Esmolol, milrinone and esmolol + milrinone reduced myocyte apoptosis rates by 22%, 37% and 60%, respectively, compared with saline (p < 0.01). CONCLUSIONS: Late-ischemia/early reperfusion therapy with esmolol + milrinone additively reduces LV-IS associated with robust activation of myocardial PKA and subsequent Akt-antiapoptotic pathway.

Reducing ischaemia/reperfusion injury through delta-opioid-regulated intrinsic cardiac adrenergic cells: adrenopeptidergic co-signalling.

AIMS: The purpose of this study was to determine whether intrinsic cardiac adrenergic (ICA) cells release calcitonin gene-related peptide (CGRP), exerting synergistic adrenopeptidergic cardioprotection. METHODS AND RESULTS: In situ hybridization coupled with immunostaining demonstrated that ICA cells exclusively expressed CGRP mRNA and co-expressed CGRP and delta-opioid receptor in human and rat left ventricular (LV) myocardium. Radioimmunoassay detected constitutive CGRP release from ICA cells in human and rat hearts. The delta-opioid agonist [D-Pen(25)]-enkephalin (DPDPE) increased CGRP release from ICA cells in denervated rat heart. In an ischaemia/reperfusion rat model, pre-ischaemic treatment with DPDPE reduced infarct size (IS) by 51 +/- 16% (P < 0.01). Co-infusion of beta(2)-adrenergic receptor (beta(2)-AR) and CGRP receptor (CGRP-R) antagonists increased IS by 62 +/- 23% (P < 0.01) compared with saline and abolished DPDPE-initiated IS reduction. Pre-treatment of ICA cell-myocyte co-culture with the beta(2)-AR/CGRP-R antagonists increased myocyte death rate by 24 +/- 4% (P < 0.01) and abolished DPDPE-initiated myocyte protection against hypoxia/reoxygenation (re-O(2)). In the ICA cell-depleted myocyte culture, DPDPE did not confer myocyte protection. Supplementing ICA cell-depleted myocyte culture with beta(2)-AR/CGRP-R agonists reduced hypoxia/re-O(2)-induced myocyte death by 24 +/- 5% (P < 0.01), simulating endogenous neurohormonal effects of ICA cells. Western blot analysis showed that DPDPE markedly increased phosphorylated myocardial Akt levels. This effect was abolished in the presence of beta(2)-AR/CGRP-R blockade. Terminal dUTP nick-end labelling staining analysis of the LV infarct zone demonstrated that DPDPE reduced myocyte apoptosis by 58 +/- 19% (P < 0.05), an effect that was eliminated in the presence of beta(2)-AR/CGRP-R blockade. Finally, echocardiography showed that DPDPE increased LV contractility in a manner dependent on beta-AR/CGRP-R stimulation. CONCLUSION: ICA cells constitute a delta-opioid-regulated adrenopeptidergic paracrine system conferring robust cardioprotection through beta(2)-AR/CGRP-R co-signalling, resulting in the activation of an anti-apoptotic pathway during ischaemia/reperfusion.

Erectile dysfunction in heart failure patients.

Chronic heart failure (HF) and erectile dysfunction (ED) are 2 highly prevalent disorders that frequently occur concomitantly. Coronary artery disease, HF, and ED share several common risk factors, including diabetes mellitus, hypertension, smoking, and dyslipidemia. Additionally, the distinct physiologic sequelae of HF create unique organic and psychologic factors contributing to ED in this patient population. Standard HF therapy with beta-receptor blockers, digoxin and thiazide diuretics may worsen sexual dysfunction owing to medication side effects. This may, in turn, lead to noncompliance in misguided efforts to retain satisfactory sexual activity, with secondary worsening of cardiac capacity. This review describes the unique aspects of ED in the HF population.

Changes in bone mineral density following treatment of osteomalacia.

Osteomalacia is characterized by defective mineralization and low bone mineral density (BMD). Clinical and biochemical improvements typically occur within a few weeks of starting treatment, though the bone mineral deficits may take longer to correct. We report a case series of 26 patients with frank osteomalacia (pseudo fractures on X-rays, elevated serum total alkaline phosphatase and parathyroid hormone, normal/low serum calcium and phosphorus, and low serum 25-hydroxy vitamin D) who were followed-up for changes in BMD during treatment using dual- energy X-ray absorptiometry (DXA). There were 23 patients with nutritional vitamin D deficiency, 2 with malabsorption syndrome, and 1 with renal tubular acidosis. All patients were treated with vitamin D and calcium; the 3 patients with associated disorders were treated accordingly. At baseline, there was low BMD at all sites tested. The rate of increase in vertebral and hip BMD was rapid in the initial few months, which subsequently slowed down. In contrast to the large increases in BMD at the femoral neck and lumbar spine, the radial BMD did not recover. At the time when most patients had marked clinical and biochemical improvement (2.8+/-1.4 mo), the vertebral and hip BMD, although improved from baseline, had not completely recovered. Bone loss at the forearm (cortical site) appears to be largely irreversible. Although the clinical correlates of these changes are presently unclear, BMD measurements are useful in assessing the initial severity of bone loss as well as the response to therapy.

Increased concentrations of lipoprotein(a), circadian rhythms and metabolic reactions evoked by acute myocardial infarction, associated with acute reactions in relation to large breakfasts.

Of 54 patients with acute coronary artery disease (CAD) that were included in this study, 41 patients had acute myocardial infarction (AMI), five patients possible MI, four patients unstable angina and the remaining four angina pectoris. The control subjects (n = 85) were randomly selected from the general population of the city of Moradabad of similar age range after exclusion for CAD (n = 9), diabetes (n = 6) and excess intake of trans fatty acids (n = 20). The incidence of lipoprotein(a) excess (> 30 mg/dl; 42.6 vs 24.7%; P < 0.05) and mean concentration of lipoprotein(a) (Lp[a], 6.4 mg/dl, 95% confidence interval: 2.8-10.5; P < 0.05) was significantly greater in the acute CAD group compared with control subjects. Mean total cholesterol and triglycerides were significantly higher and mean nitrite level lower in the study group as compared with the control group.There was a significant greater incidence of cardiac events in the second quarter of the day compared with the fourth quarter. Lp(a), triglycerides, blood glucose, plasma insulin, malondialdehyde, diene conjugates, TBARS and TNF-alpha and IL-6 levels, which were significantly greater during the acute phase, showed a significant decline and serum nitrite and coenzyme Q demonstrated an increase at 4 weeks of follow-up when the acute reactions evoked by MI had been controlled. Large breakfasts were a predisposing factor for cardiac events in the second quarter of the day and it was significantly associated with metabolic reactions. The findings indicate that acute reactions as a result or as circadian rhythms appear to be important in the pathogenesis of AMI-associated complications and that a large breakfast in association with nitrite deficiency may further trigger the circadian rhythms. However, more studies in a larger number of subjects would be necessary in order to confirm our findings.

Mechanisms of acute myocardial infarction study (MAMIS).

Acute myocardial infarction (AMI) is a highly dynamic event, which is associated with marked neuroendocrinological dysfunction in addition to cardiac damage. The immediate trigger for AMI is not precisely known. Studies conducted by Lown, Braunwald, Halberg, Otsuka and our group have demonstrated a marked increase in sympathetic activity, oxidative stress, and magnesium and potassium deficiency during AMI. Clinical studies have reported an increased incidence of AMI, sudden death and ischemia during first quarter of the day when there is a rapid withdrawal of vagal activity and increase in sympathetic tone. In one case-control study of 202 patients with AMI, there was a significant (P < 0.02) increase in cardiac events in the second quarter of the day compared to other quarters, respectively (16.8%, 41.0%, 13.8%, 28.2% per quarter). This characteristic remained prevalent in both men and women and among patients with and without known AMI (n = 52), diabetes (n = 53) or hypertension (n = 75). Triggers of AMI were noted among 162 (82.2%) of the patients. Neuropsychological mechanisms were observed as follows: emotional stress (45.5%), sleep deprivation (27.7%), cold climate (29.2%), hot climate (24.7%), large meals (47.5%) and physical exertion (31.2%). These triggering factors are known to enhance sympathetic activity and decrease vagal tone, resulting in an increased secretion of plasma cortisol, noradrenaline, aldosterone, angiotension-converting enzyme (ACE), interleukin (IL)-1, -2, -6, -18, and tumor necrosis factor-alpha (TNF-alpha), all of which are are proinflammatory agents. There is also a deficiency in the serum levels of vitamin A, E, and C and magnesium, potassium, melatonin, and IL-10 (an anti-inflammatory agent). In our study, we found a decrease in magnesium, potassium, vitamin A, E, C and beta carotene combined with an increase in thiobarbituric acid-reactive substances (TBARS), MDA and diene conjugates, TNF-alpha and IL-6, all of which are indicators of oxidative damage and proinflammatory activity, respectively.