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A device for measuring biological small volume liquid samples in real time is appealing. One way to achieve this is by using a microwave sensor based on reflection measurement. A prototype sensor was manufactured from low cost printed circuit board (PCB) combined with a microfluidic channel made of polymethylsiloxane (PDMS). Such a sensor was simulated, manufactured, and tested including a vacuum powered sample delivery system with robust fluidic ports. The sensor had a broad frequency band from 150 kHz to 6 GHz with three resonance frequencies applied in sensing. As a proof of concept, the sensor was able to detect a NaCl content of 125 to 155 mmol in water, which is the typical concentration in healthy human blood plasma.

The MYC-like sequence CATGTG plays an important role in the dehydration-inducible expression of the Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION STRESS 1 (ERD1) gene, which encodes a ClpA (ATP binding subunit of the caseinolytic ATP-dependent protease) homologous protein. Using the yeast one-hybrid system, we isolated three cDNA clones encoding proteins that bind to the 63-bp promoter region of erd1, which contains the CATGTG motif. These three cDNA clones encode proteins named ANAC019, ANAC055, and ANAC072, which belong to the NAC transcription factor family. The NAC proteins bound specifically to the CATGTG motif both in vitro and in vivo and activated the transcription of a beta-glucuronidase (GUS) reporter gene driven by the 63-bp region containing the CATGTG motif in Arabidopsis T87 protoplasts. The expression of ANAC019, ANAC055, and ANAC072 was induced by drought, high salinity, and abscisic acid. A histochemical assay using P(NAC)-GUS fusion constructs showed that expression of the GUS reporter gene was locafized mainly to the leaves of transgenic Arabidopsis plants. Using the yeast one-hybrid system, we determined the complete NAC recognition sequence, containing CATGT and harboring CACG as the core DNA binding site. Microarray analysis of transgenic plants overexpressing either ANAC019, ANAC055, or ANAC072 revealed that several stress-inducible genes were upregulated in the transgenic plants, and the plants showed significantly increased drought tolerance. However, erd1 was not upregulated in the transgenic plants. Other interacting factors may be necessary for the induction of erd1 in Arabidopsis under stress conditions.

Purpose Flotation methods are widely used to detect oocysts/cysts of protozoans and eggs of helminths, except trematodes. However, details regarding the concentration and recovery rates of these parasites are poorly understood. Methods Using Eimeria tenella oocysts as a model parasite, the present study evaluated three check points: (1) the proportion of parasites that remain floating in flotation solution (sucrose or saturated saline) during centrifugation, (2) the proportion of oocysts that naturally float after addition of flotation solution after centrifugation, and (3) the rate of recovery on cover slips after completion of the flotation protocol. Results After centrifugation in sucrose solution and saturated saline solution, 82.4% and 60.3% of oocysts floated, respectively. After addition of flotation solution after the final centrifugation step, the recovery rates for oocysts that naturally floated again for 30 min in sucrose and saturated saline were 39.2% and 38.2%, respectively. The recovery rate on cover slips as the final step after performing a commonly used flotation method was 36.4% in sucrose solution (the rate for saturated saline solution could not be assessed due to rapid crystallization). Conclusion Our results suggest that floating oocysts could have become dispersed by the addition of flotation solution, and not all of these oocysts remained floating after an additional 30 min of settling time although collection on cover slips could be effective for accurate recovery.

Objectives 1- To compare the effectiveness of 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline and a novel solution Neem extract ( Azardirachta indica ) in reducing intra-oral viral load in COVID-19 positive patients. 2- To determine the salivary cytokine profiles of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL- 17 among COVID-19 patients subjected to 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline or Neem extract ( Azardirachta indica) based gargles. Trial design This will be a parallel group, quadruple blind-randomised controlled pilot trial with an add on laboratory based study. Participants A non-probability, purposive sampling technique will be followed to identify participants for this study. The clinical trial will be carried out at the Aga Khan University Hospital (AKUH), Karachi, Pakistan. The viral PCR tests will be done at main AKUH clinical laboratories whereas the immunological tests (cytokine analysis) will be done at the Juma research laboratory of AKUH. The inclusion criteria are laboratory-confirmed COVID-19 positive patients, male or female, in the age range of 18-65 years, with mild to moderate disease, already admitted to the AKUH. Subjects with low Glasgow coma score, with a history of radiotherapy or chemotherapy, who are more than 7 days past the onset of COVID- 19 symptoms, or intubated or edentulous patients will be excluded. Patients who are being treated with any form of oral or parenteral antiviral therapy will be excluded, as well as patients with known pre-existing chronic mucosal lesions such as lichen planus. Intervention and comparator Group A (n=10) patients on 10 ml gargle and nasal lavage using 0.2% Povidone-Iodine (Betadiene® by Aviro Health Inc./ Pyodine® by Brooks Pharma Inc.) for 20-30 seconds, thrice daily for 6 days. Group B (n=10) patients will be subjected to 10 ml gargle and nasal lavage using 1% Hydrogen peroxide (HP® by Karachi Chemicals Products Inc./ ActiveOxy® by Boumatic Inc.) for 20-30 seconds, thrice daily for 6 days. Group C will comprised of (n=10) subjects on 10ml gargle and nasal lavage using Neem extract solution ( Azardirachta indica ) formulated by Karachi University (chemistry department laboratories) for 20-30 seconds, thrice daily for 6 days. Group D (n=10) patients will use 2% hypertonic saline (Plabottle® by Otsuka Inc.) gargle and nasal lavage for a similar time period. Group E (n=10) will serve as positive controls. These will be given simple distilled water gargles and nasal lavage for 20-30 seconds, thrice daily for six days. For nasal lavage, a special douche syringe will be provided to each participant. Its use will be thoroughly explained by the data collection officer. After each use, the patient is asked not to eat, drink, or rinse their mouth for the next 30 minutes. Main outcomes The primary outcome is the reduction in the intra-oral viral load confirmed with real time quantitative PCR. Randomisation The assignment to the study group/ allocation will be done using the sealed envelope method under the supervision of Clinical Trial Unit (CTU) of Aga Khan University, Karachi, Pakistan. The patients will be randomised to their respective study group (1:1:1:1:1 allocation ratio) immediately after the eligibility assessment and consent administration is done. Blinding (masking) The study will be quadruple-blinded. Patients, intervention provider, outcome assessor and the data collection officer will be blinded. The groups will be labelled as A, B, C, D or E. The codes of the intervention will be kept in lock & key at the CTU and will only be revealed at the end of study or if the study is terminated prematurely. Numbers to be randomised (sample size) As there is no prior work on this research question, so no assumptions for the sample size calculation could be made. The present study will serve as a pilot trial. We intend to study 50 patients in five study groups with 10 patients in each study group. For details, please refer to Fig.  1 for details. Trial Status Protocol version is 7.0, approved by the department and institutional ethics committees and clinical trial unit of the university hospital. Recruitment is planned to start as soon as the funding is sanctioned. The total duration of the study is expected to be 6 months i.e. August 2020-January 2021. Trial registration This study protocol was registered at www.clinicaltrials.gov on 10 April 2020 NCT04341688 . Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1 ). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2 ). Fig. 1 Flow diagram of study-participants’ timeline

Background Current international guidelines recommend the use of a daily topical exit-site antimicrobial to prevent peritoneal dialysis (PD)-related infections. Although nonantibiotic-based therapies are appealing because they may limit antimicrobial resistance, no controlled trials have been conducted to compare topical antimicrobial agents with usual exit-site care for the prevention of PD-related infections among the Thai PD population. We propose a controlled three-arm trial to examine the efficacy and safety of a daily chlorhexidine gluconate-impregnated patch versus mupirocin ointment versus usual exit-site care with normal saline for the prevention of PD-related infections. Methods/Designs This study is a randomized, double-blind, multicenter, active-controlled, clinical trial. Adult patients aged 18 years or older who have end-stage kidney disease and are undergoing PD will be enrolled at three PD Centers in Thailand. A total of 354 PD patients will be randomly assigned to either the 2% chlorhexidine gluconate-impregnated patch, mupirocin ointment, or usual exit-site care with normal saline dressing according to a computer-generated random allocation sequence. Participants will be followed until discontinuation of PD or completion of 24 months. The primary study outcomes are time to first PD-related infection (exit-site/tunnel infection or peritonitis) event and the overall difference in PD-related infection rates between study arms. Secondary study outcomes will include (i) the rate of infection-related catheter removal and PD technique failure, (ii) rate of nasal and exit-site Staphylococcus aureus colonization, (iii) healthcare costs, and (iv) skin reactions and adverse events. We plan to conduct a cost-utility analysis alongside the trial from the perspectives of patients and society. A Markov simulation model will be used to estimate the total cost and health outcome in terms of quality-adjusted life years (QALYs) over a 20-year time horizon. An incremental cost-effectiveness ratio in Thai Baht and U.S. dollars per QALYs gained will be illustrated. A series of probabilistic sensitivity analyses will be conducted to assess the robustness of the cost-utility analysis findings. Discussion The results from this study will provide new clinical and cost-effectiveness evidence to support the best strategy for the prevention of PD-related infections among the Thai PD population. Trial registration ClinicalTrials.gov, NCT02547103 . Registered on September 11, 2015.

Objectives: The aim of the study was to compare the diagnostic yield and safety profile of sputum induction (SI) with nebulized racemic salbutamol solution versus hypertonic saline in smear-negative pulmonary tuberculosis (TB). Methods: The prospective study was conducted at Songklanagarind Hospital, Thailand. Suspected smear-negative pulmonary TB cases were recruited and randomized to receive SI with either nebulized racemic salbutamol solution or 3% sodium chloride (NaCl) solution. Induced sputum was examined with the acid-fast bacilli (AFB) smear test and cultured for Mycobacterium tuberculosis. The efficacy and adverse events of SI were analyzed. Results: A total of 59 patients received SI with nebulized racemic salbutamol solution and 53 received 3% NaCl solution. There was no significant difference between the two groups in the average quantity of induced sputum (1.3 ± 0.1 versus 1.2 ± 0.2 ml, p = 0.5). The percentages of positive AFB smear and TB cultures in the salbutamol group were 15% and 22%, and 13% and 17% in the 3% NaCl group (p = 0.5), respectively. Racemic salbutamol solution could increase the TB diagnostic yield similarly to 3% NaCl, but incurred less chest tightness (5% versus 15%) and bronchospasm (0% versus 11.3%, p = 0.02) compared with 3% NaCl. Conclusions: SI by nebulized racemic salbutamol solution offers equal benefits to 3% NaCl solution in increasing both sputum quantity and diagnostic yield in smear-negative patients suspected of having pulmonary TB. Nebulized racemic salbutamol does not produce bronchospasm and chest tightness occurs less frequently than with 3% NaCl. Therefore, SI with nebulized racemic salbutamol solution should be considered as a good alternative noninvasive diagnostic tool for the diagnosis of pulmonary TB when hypertonic saline is unavailable or contraindicated.

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.

Proper DNA extraction is an essential step in molecular biology research, for various downstream applications. Several modifications have been made to the first extraction protocol by Friedrich Miescher in 1869. The current work aimed to standardize an eco-friendly and quicker DNA extraction process that could be used in resource-limited laboratories by utilizing low-priced household liquid detergents and easily accessible salt. The pectoral fin tissues were lysed at 58°C with two modified lysis buffers using detergent 1 & 2 along with the conventional lysis buffer (SDS) as control. Instead of extraction with organic solvents, a 5M edible salt solution was used. This modified protocol resulted in yielding 3269.67 (±108.7) ng/µl and 3000 (± 15) ng/µl of DNA using detergent 1 and 2 with comparable quality of DNA as confirmed by OD 260/280 , i.e., 1.7 (± 0.026) and 1.72 (± 0.015) respectively, while the conventional method gave a maximum of 2512.33 (± 45.78) ng/µl of DNA with 1.76 (± 0.021) OD 260/280 values. The overall cost of the proposed protocol was found almost 32 times less than the conventional method. The quality of DNA obtained by the modified protocol was pure enough to be used in PCR amplification of both nuclear (microsatellite) and mitochondrial ( COX1 ) DNA for further application of genotyping. This modified protocol for DNA extraction from fish fin was faster (half the time required than the SDS lysis), of comparable quality and even better quantity with significantly lesser overall cost, and can be recommended for DNA extraction from fish samples in any resource-constrained laboratories.

Traditional fermented horsemeat and horsemeat sausages in Xinjiang are rich in microbial resources, mainly including lactic acid bacteria and yeasts. Although the probiotic effects of lactic acid bacteria have been widely studied, there is relatively little research on the probiotic properties of yeasts, which limits their application in functional food development. This study isolated 44 yeast strains from traditional fermented horsemeat products in Xinjiang, and 16 strains exhibiting strong tolerance to temperature, acidic pH, simulated gastrointestinal fluids, and high bile salt solution were screened. Molecular identification revealed that these strains belonged to Candida zeylanoides (C-5, C-8, C-13, C-15, C-21, C-22, C-24, R-8), Candida parapsilosis (R-10, R-12, R-14, R-19, R-20), Candida metapsilosis (R-18), and Rhodotorula alborubescens (R-6, R-11). Several strains demonstrated significantly higher cell surface hydrophobicity, auto-aggregation, and co-aggregation with pathogenic bacteria than the control strain Saccharomyces boulardii CNCM I-745, indicating strong intestinal adhesion and antagonistic potential. All strains have high antioxidant activity (> 60%) and certain cholesterol-lowering ability. Furthermore, extracellular metabolites from select strains exhibited antibacterial activity and potential glucose-lowering effects. Specifically, C. zeylanoides C-8 inhibited α-glucosidase by 54.14%, and C-8 inhibited angiotensin-converting enzyme (ACE) by 37.05%. Hemolysis assays confirmed that all strains were non-hemolytic, suggesting good biosafety. These findings highlight the multi-functional probiotic potential of yeast strains from traditional fermented horsemeat products and provide a scientific basis for their development as novel functional food ingredients.

To minimize sample dilution effect on SARS-CoV-2 pool testing, we assessed analytical and diagnostic performance of a new methodology, namely swab pooling. In this method, swabs are pooled at the time of collection, as opposed to pooling of equal volumes from individually collected samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individuals. Having individual testing as reference, no false-positives or false-negatives were observed for swab pooling. In additional 18,922 patients screened with swab pooling (1,344 pools), mean Cq differences between individual and pool samples ranged from 0.1 (Cr.I. -0.98 to 1.17) to 2.09 (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic patients were screened using 4,400 RT-qPCR assays. This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.