IU Ml-1 Research Articles

Dried Blood Matrix as a New Material for the Detection of DNA Viruses.

The gold standard for diagnosing viruses such as the Hepatitis B Virus has remained largely unchanged, relying on conventional methods involving extraction, purification, and polymerase chain reaction (PCR). This approach is hindered by limited availability, as it is time-consuming and requires highly trained personnel. Moreover, it suffers from low recovery rates of the nucleic acid molecules for samples with low copy numbers. To address the challenges of complex instrumentation and low recovery rate of DNA, a drying process coupled with thermal treatment of whole blood is employed, resulting in the creation of a dried blood matrix characterized by a porous structure with a high surface-to-volume ratio where it also inactivates the amplification inhibitors present in whole blood. Drawing on insights from Brunauer-Emmett-Teller (BET)- Barrett-Joyner-Halenda (BJH) analysis, scanning electron microscopy (SEM), and fluorescence recovery after photobleaching (FRAP), detection assay is devised for HBV, as a demonstration, from whole blood with high recovery of DNA and simplified instrumentation achieving a limit of detection (LOD) of 10 IU mL-1. This assay can be completed in <1.5 h using a simple heater, can be applied to other DNA viruses, and is expected to be suitable for point-of-care, especially in low-resource settings.

Extraction of cellulose from halophytic plants for the synthesis of a novel biocomposite.

Cellulose nanofibers, a sustainable and promising material with widespread applications, exhibit appreciable strength and excellent mechanical and physicochemical properties. The preparation of cellulosic nanofibers from food or agricultural residue is not sustainable. Therefore, this study was designed to use three halophytic plants (Cressa cretica, Phragmites karka, and Suaeda fruticosa) to extract cellulose for the subsequent conversion to cellulosic nanofibers composites. The other extracted biomass components including lignin, hemicellulose, and pectin were also utilized to obtain industrially valuable enzymes. The maximum pectinase (31.56 IU mL-1), xylanase (35.21 IU mL-1), and laccase (15.89 IU mL-1) were produced after the fermentation of extracted pectin, hemicellulose, and lignin from S. fruticosa, P. karka, and C. cretica, respectively. Cellulose was methylated (with a degree of substitution of 2.4) and subsequently converted into a composite using polyvinyl alcohol. Scanning electron microscopy and Fourier-transform infrared spectroscopy confirmed the successful synthesis of the composites. The composites made up of cellulose from C. cretica and S. fruticosa had a high tensile strength (21.5 and 15.2 MPa) and low biodegradability (47.58% and 44.56%, respectively) after dumping for 3 months in soil, as compared with the composite from P. karka (98.79% biodegradability and 4.9 MPa tensile strength). Moreover, all the composites exhibited antibacterial activity against gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) and gram-positive bacteria (Staphylococcus aureus). Hence, this study emphasizes the possibility for various industrial applications of biomass from halophytic plants.

Plant growth-promoting and biocontrol traits of endophytic Bacillus licheniformis against soft rot causing Pythium myriotylum in ginger plant.

Bacterial endophytes from plants harbor diverse metabolites that play major roles in biocontrol and improve plant growth. In this study, a total of 12 endophytic bacteria were isolated from the ginger rhizome. The strain K3 was highly effective in preventing mycelia growth of Pythium myriotylum (78.5 ± 1.5% inhibition) in dual culture. The cell-free extract (2.5%) of endophyte K3 inhibited 76.3 ± 4.8% mycelia growth, and 92.4 ± 4.2% inhibition was observed at a 5% sample concentration. The secondary metabolites produced by Bacillus licheniformis K3 showed maximum activity against Pseudomonas syringae (24 ± 1 mm zone of inhibition) and Xanthomonas campestris (28 ± 3 mm zone of inhibition). The strain K3 produced 28.3 ± 1.7 IU mL-1 protease, 28.3 ± 1.7 IU mL-1 cellulase, and 2.04 ± 0.13 IU mL-1 chitinase, respectively. The ginger rhizome treated with K3 in the greenhouse registered 53.8 ± 1.4% soft rot incidence, and the streptomycin-treated pot registered 78.3 ± 1.7% disease incidence. The selected endophyte K3 improved ascorbate peroxidase (1.37 ± 0.009 µmole ASC min-1 mg-1 protein), catalase (8.7 ± 0.28 µmole min-1 mg-1 protein), and phenylalanine ammonia-lyase (26.2 ± 0.99 Umg-1) in the greenhouse. In addition, K3 treatment in the field trial improved rhizome yield (730 ± 18.4 g) after 180 days (p < 0.01). The shoot length was 46 ± 8.3 cm in K3-treated plants, and it was about 31% higher than the control treatment (p < 0.01). The lytic enzyme-producing and growth-promoting endophyte is useful in sustainable crop production through the management of biotic stress.

A Dual-domain Engineered Antibody for Efficient HBV Suppression and Immune Responses Restoration.

Chronic hepatitis B (CHB) remains a major public health concern because of the inefficiency of currently approved therapies in clearing the hepatitis B surface antigen (HBsAg). Antibody-based regimens have demonstrated potency regarding virus neutralization and HBsAg clearance. However, high dosages or frequent dosing are required for virologic control. In this study, a dual-domain-engineered anti-hepatitis B virus (HBV) therapeutic antibody 73-DY is developed that exhibits significantly improved efficacy regarding both serum and intrahepatic viral clearance. In HBV-tolerant mice, administration of a single dose of 73-DY at 2mgkg-1 is sufficient to reduce serum HBsAg by over 3 log10 IU mL-1 and suppress HBsAg to < 100 IU mL-1 for two weeks, demonstrating a dose-lowering advantage of at least tenfold. Furthermore, 10mgkg-1 of 73-DY sustainably suppressed serum viral levels to undetectable levels for ≈ 2 weeks. Molecular analyses indicate that the improved efficacy exhibited by 73-DY is attributable to the synergy between fragment antigen binding (Fab) and fragment crystallizable (Fc) engineering, which conferred sustained viral suppression and robust viral eradication, respectively. Long-term immunotherapy with reverse chimeric 73-DY facilitated the restoration of anti-HBV immune responses. This study provides a foundation for the development of next-generation antibody-based CHB therapies.

Stability of BK polyomavirus DNA in urine over time and analytical evaluation of an automated BKV quantitative nucleic acid test.

Introduction. BK polyomavirus (BKPyV) quantitative testing is an important screening tool post-transplantation, although interpretation can be challenging due to lack of standardization, assay heterogeneity and variability of BKPyV DNA over time (in urine).Methods. Remnant clinical EDTA plasma and urine samples were tested by the cobas BKV test and a validated laboratory-developed test (LDT). Accuracy [positive and negative percent agreement (PPA and NPA), Pearson's correlation, Bland-Altman analysis] and reproducibility were evaluated. To assess BKPyV DNA stability in urine, prospective urine samples were maintained at two different storage temperatures and tested in triplicate over 7 days.Results. Overall PPA was 95.6 % (43/45) and NPA was 94.4 % (170/180). For plasma, Pearson's correlation (0.950) and Bland-Altman analysis (0.113±0.22 log10 IU ml-1) showed high agreement. For neat urine, Pearson's correlation (0.842) and Bland-Altman analysis (0.326±0.80 log10 IU ml-1) showed somewhat higher variability. Reproducibility was high for the cobas BKV versus the LDT. BKPyV DNA levels in neat urine remained relatively stable over 7 days at both storage temperatures, although outlier results were intermittently detected.Conclusion. The cobas BKV test showed high agreement and reproducibility compared to the reference LDT. BKPyV viral load testing in urine has known limitations, but neat urine can be processed by the cobas BKV.

Association between ABO and Rh blood groups and Toxoplasma gondii infection: A cross sectional study.

We aimed to determine the association between the seropositivity to Toxoplasma gondii and the ABO and Rh blood groups in 2,053 people. ABO and Rhesus blood groups and anti-T. gondii IgG and IgM antibodies were determined using commercially available assays. Of the 2,053 people studied, 171(8.3%) were positive for anti-T. gondii IgG antibodies. Sixty-five (38.0%) and 36 (21.1%) of these 171 individuals had high anti-T. gondii IgG antibody levels (≥150 IU mL-1) and anti-T. gondii IgM antibodies, respectively. We found the following prevalences of T. gondii infection among the ABO groups: 8.5% in group A, 4.3% in group B, 4.7% in group AB, and 8.9% in group O (P = 0.19). The prevalences of T. gondii infection among Rh groups were: 8.4% in the Rh-positive group and 7.1% in theRh-negative group (P = 0.58). Logistic regression analysis showed that the frequencies of ABO andRh blood groups were similar (P > 0.05) among people with positive and negative serology for anti-T. gondii IgG antibodies, with high (≥150 IU mL-1) and lower (<150 IU mL-1) levels of anti-T.gondii IgG antibodies, and with positive and negative serology for anti-T. gondii IgM antibodies. Results does not support an association between T. gondii infection and ABO and Rh blood groups.

Multiplexed electrical detection of whole viruses from plasma in a microfluidic platform.

The advancement of point-of-care diagnostics is crucial to improving patient outcomes, especially in areas with low access to hospitals or specialized laboratories. In particular, rapid, sensitive, and multiplexed detection of disease biomarkers has great potential to achieve accurate diagnosis and inform high quality care for patients. Our Coulter counting and immunocapture based detection system has previously shown its broad applicability in the detection of cells, proteins, and nucleic acids. This paper expands the capability of the platform by demonstrating multiplexed detection of whole-virus particles using electrically distinguishable hydrogel beads by demonstrating the capability of our platform to achieve simultaneous detection at clinically relevant concentrations of hepatitis A virus (>2 × 103 IU mL-1) and human parvovirus B19 virus like particles (>106 IU mL-1) from plasma samples. The expanded versatility of the differential electrical counting platform allows for more robust and diverse testing capabilities.

Integrated device for plasma separation and nucleic acid extraction from whole blood toward point-of-care detection of bloodborne pathogens.

Sample preparation presents a major challenge in point-of-care (POC) diagnostic assays, including ones requiring whole blood as the starting specimen. This study presents an integrated sample preparation device - which we call PRECISE - that performs both plasma separation and nucleic acid extraction, enabling streamlined sample preparation from whole blood requiring only a commercially available blood collection tool and a syringe, and no other external equipment or electricity. Plasma separation is performed using a dual-membrane filter (which filters out blood components while limiting membrane clogging) integrated into the cartridge, and nucleic acid extraction is performed by users moving magnets (to mix the samples, and along a guided track). The plasma filtration demonstrated recovery on par with lab-based centrifugation, and the extraction module showed performance similar to benchtop-based magnetic bead extraction. A sample-to-result demonstration on 50 μL of whole blood spiked with virions of hepatitis C virus (HCV), operating the PRECISE cartridge in 16 minutes followed by benchtop PCR, showed a limit of detection (∼6770 IU mL-1) on the order of the minimal requirements of target product profile for POC HCV detection. Future work on the PRECISE cartridge, building on POC accessibility and fast sample preparation demonstrated in this work, may enable detection of bloodborne pathogens from whole-blood specimens collected at the POC.

A therapeutic dose and its pharmacokinetics of ropeginterferon Alfa-2b for hepatitis C treatment

Ropeginterferon alfa-2b is a novel mono-pegylated proline-interferon. Its biweekly dosing schema has demonstrated tolerability and clinical efficacy for treating chronic hepatitis in previous clinical studies. This trial evaluates the pharmacokinetics of 400μg ropeginterferon alfa-2b in patients with chronic hepatitis C virus (HCV) and provides the data to support the clinical utility of ropeginterferon alfa-2b at 400 μg. Seventeen patients with chronic HCV genotype 2 were enrolled to receive a single injection of 400μg ropeginterferon alfa-2b plus 14-day treatment of ribavirin. Pharmacokinetics, safety, and HCV RNA reduction/clearance were assessed. Tmax was 154.003h and T1/2 was 114.273h. The Cmax was 29.823ngmL-1. AUClast was 9364.292h∗ng mL-1 and AUCinf was 11084.317h∗ng mL-1. All adverse events were mild or moderate, and there were no serious adverse events. A 1000-fold reduction in the geometric mean of HCV RNA was observed 14d after the single injection of ropeginterferon alfa-2b. Two patients achieved clearance of HCV RNA, and the other five patients had HCV RNA levels lower than 200 IU mL-1. Ropeginterferon alfa-2b at 400μg led to PK exposures associated with safety and notable clinical activity in patients with chronic HCV. This study suggests that ropeginterferon alfa-2b at 400μg is an acceptable dosing regimen for treating chronic HCV and also provides supporting data for the clinical use of ropeginterferon alfa-2b at a higher starting dose for other indications.

Utilization of wild Cressa cretica biomass for pectinase production from a halo-thermotolerant bacterium.

Halophytes are the native inhabitants of saline environment. Their biomass can be considered as a potential substrate for the production of microbial enzymes. This study was intended at feasible utilization of a halophytic biomass, Cressia cretica, for pectinase production using a halo- and thermo-tolerant bacterium, Bacillus vallismortis MH 10. The data from fractionation of the C. cretica biomass revealed presence of 17% pectin in this wild biomass. Seven different factors (temperature, agitation, pH, inoculum size, peptone concentration, substrate concentration and incubation time) affecting pectinase production using C. cretica were assessed through a statistical tool, Plackett-Burman design. Consequently, two significant factors (incubation time and peptone concentration) were optimized using the Central Composite design. The strain produced 20 IU mL-1 of pectinase after 24h under optimized conditions. The enzyme production kinetics data also confirmed that 24h is the most suitable cultivation period for pectinase production. Fourier transform infrared spectroscopy and scanning electron microscopy of C. cretica biomass ascertained utilization of pectin and structural changes after fermentation. The purification of pectinase by using DEAE column yielded specific activity and purification fold of 88.26 IU mg-1 and 3.2, respectively. The purified pectinase had a molecular weight of>65kDa. This study offers prospects of large-scale production of pectinase by halotolerant strain in the presence of economical and locally grown substrate that makes the enzyme valuable for various industrial operations. This article is protected by copyright. All rights reserved.

Evaluation and validation of a commercial ELISA versus the in vitro toxin neutralization assay for determination of diphtheria anti-toxin in human serum.

Introduction. Diphtheria is a potentially life-threatening infection and remains endemic in many low- and middle-income countries (LMICs). A reliable, low-cost method for serosurveys in LMICs is warranted to estimate the accurate population immunity to control diphtheria.Hypothesis/Gap Statement. The correlation between the ELISA results against diphtheria toxoid and the gold standard diphtheria toxin neutralization test (TNT) values is poor when ELISA values are <0.1 IU ml-1, which results in inaccurate estimates of susceptibility in populations when ELISA is used for measuring antibody levels.Aim. To explore methods to accurately predict population immunity and TNT-derived anti-toxin titres from ELISA anti-toxoid results.Methodology. A total of 96 paired serum and dried blood spot (DBS) samples collected in Vietnam were used for comparison of TNT and ELISA. The diagnostic accuracy of ELISA measurement with reference to TNT was assessed by area under the receiver operating characteristic (ROC) curve (AUC) and other parameters. Optimal ELISA cut-off values corresponding to TNT cut-off values of 0.01 and 0.1 IU ml-1 were identified by ROC analysis. A method based on the multiple imputation approach was also applied to estimate TNT measurements in a dataset that only included ELISA results. These two approaches were then applied to ELISA results previously generated from 510 subjects in a serosurvey in Vietnam.Results. The ELISA results on DBS samples showed a good diagnostic performance compared to TNT. The cut-off values for ELISA measurement corresponding to the TNT cut-off values of 0.01 IU ml-1 were 0.060 IU ml-1 in serum samples, and 0.044 IU ml-1 in DBS samples. When a cut-off value of 0.06 IU ml-1 was applied to the 510 subject serosurvey data, 54 % of the population were considered susceptible (<0.01 IU ml-1). The multiple imputation-based approach estimated that 35 % of the population were susceptible. These proportions were much larger than the susceptible proportion estimated by the original ELISA measurements.Conclusion. Testing a subset of sera by TNT combined with ROC analysis or a multiple imputation approach helps to adjust ELISA thresholds or values to assess population susceptibility more accurately. DBS is an effective low-cost alternative to serum for future serological studies for diphtheria.

Biological pretreatment of sugarcane bagasse for the production of fungal laccase and bacterial cellulase.

Sugarcane bagasse (SB) is a promising source of appreciable quantities of fermentable sugars. However, the presence of lignin hinders utilization of these carbohydrates and hence pretreatment to remove lignin is necessarily carried out. Here, a biological pretreatment method was synchronized with the production of a thermostable cellulase using SB as a raw material. Initially, bagasse was fermented by a laccase producing fungus, Trametes pubescens MB 89 under solid state fermentation (SSF) and a titer of 1758 IU mL-1 of laccase was obtained. Investigations of nine factors affecting laccase production through Plackett Burman design improved the titers to 6539 IU mL-1 . Five factors (incubation period, concentration of CuSO4 , temperature, moisture content, and particle size) were found significant which were optimized through Central Composite design leading to an improvement in the titers by ~5 folds (8841 IU mL-1 ). Biologically pretreated SB was fermented by a thermophilic bacterium, Neobacillus sedimentimangrovi UE25, that yielded 8.64 IU mL-1 of cellulase. Delignification and cellulose utilization were affirmed by structural analysis through FTIR and SEM. The synchronized process yielded higher titers of laccase and cellulase under SSF of SB with the minimum use of corrosive chemicals.

Utilization of Corncob as an Immobilization Matrix for a Xylanolytic Yeast Strain.

Immobilization of microbial cells for the production of industrially important enzymes has been reported to offer the advantages of recyclability, higher yields and cost effectiveness. The search for an appropriate matrix that is affordable and easy to prepare is a significant topic in microbial biotechnology. Here, an abundant type of agro-industrial waste-corncob-was utilized as an immobilization matrix for the production of xylanase from an indigenous yeast strain, Saccharomyces cerevisiae MK-157. This is the first report describing xylanase production from immobilized S. cerevisiae. To render the corncob matrix more porous, alkaline pretreatment was undertaken and yeast cells were immobilized on the matrix by cultivating at 30 °C for 48 h in Sabouraud dextrose broth. After incubation, the immobilized matrix was transferred to mineral salt medium containing 1% xylan and incubated at 30 °C for 24 h. Xylanase production was determined in cell-free culture supernatant and the matrix was recycled for up to seven cycles. Moreover, xylanase-mediated saccharification was carried out using sugarcane bagasse as a substrate and the release of reducing sugars was monitored. The results showed that the immobilized yeast produced 4.97 IU mL-1 xylanase in the first production cycle, indicating a >tenfold increase compared to the free cells. Xylanase production further increased to its maximum levels (9.23 IU mL-1) in the fourth production cycle. Nonetheless, the cells retained 100% productivity for up to seven cycles. The volumetric and specific productivity of xylanase were also the highest in the fourth cycle. Scanning electron microscopy images revealed the rough surface of the untreated corncob, which became more porous after alkaline pretreatment. Immobilized yeast cells were also visible on the corncob pieces. The saccharification of a natural resource-sugarcane bagasse-using xylanase preparation yielded 26 mg L-1 of reducing sugars. Therefore, it can be concluded that yeast strains can yield sufficient quantities of xylanase, allowing possible biotechnological applications. Moreover, corncob can serve as a cost-effective matrix for industrially important yeast strains.

Engineered Protein Copolymers for Heparin Neutralization and Detection.

Heparin is a widely applied anticoagulant agent. However, in clinical practice, it is of vital importance to reverse its anticoagulant effect to restore the blood-clotting cascade and circumvent side effects. Inspired by protein cages that can encapsulate and protect their cargo from surroundings, we utilize three designed protein copolymers to sequester heparin into inert nanoparticles. In our design, a silk-like sequence provides cooperativity between proteins, generating a multivalency effect that enhances the heparin-binding ability. Protein copolymers complex heparin into well-defined nanoparticles with diameters below 200 nm. We also develop a competitive fluorescent switch-on assay for heparin detection, with a detection limit of 0.01 IU mL-1 in plasma that is significantly below the therapeutic range (0.2-8 IU mL-1). Moreover, moderate cytocompatibility is demonstrated by in vitro cell studies. Therefore, such engineered protein copolymers present a promising alternative for neutralizing and sensing heparin, but further optimization is required for in vivo applications.

Incorporation of nisin and oregano essential oil in cow’s milk to improve the quality of fresh cheese

Firstly, this study aimed to determine the effect of adding nisin (0, 250 and 500 IU mL-1) and oregano essential oil (OEO) (0, 0.025% and 0.05%), alone or in combination, on Staphylococcus aureus load in cow’s milk. Next, it was evaluated the sensory, physicochemical and microbiological quality of fresh cheese (FC) manufactured with milk added of nisin + OEO (C1) or nisin alone (C2) compared to a control cheese (C0, milk without the addition of nisin or OEO). Results showed that adding nisin effectively reduced the S. aureus count in milk in a similar manner to adding way the combination of nisin + OEO compared to the control. FC quality evaluation results revealed that C1 and C2 did not alter the physicochemical quality of FC (pH, fat and total solids content) during storage (0-8 days) compared to the control. Furthermore, C1 and C2 reduced in a similar manner aerobic mesophilic bacteria and Enterobacteriaceae count in FC compared to the control during storage. Sensory evaluation showed that consumers did not value FC added of nisin + OEO (C1) due to its intense aroma of oregano, rating it with the lowest global quality.

Rapid vertical flow technique for the highly sensitive detection of Brucella antibodies with Prussian blue nanoparticle labeling and nanozyme-catalyzed signal amplification.

Brucellosis is a chronic infectious disease caused by Brucella, which is characterized by inflammation of reproductive organs and fetal membranes, abortion, infertility, and local inflammatory lesions of various tissues. Due to the widespread prevalence and spread of brucellosis, it has not only caused huge losses to animal husbandry, but also brought serious impacts on human health and safety. Therefore, rapid and accurate diagnosis is of great significance for the effective control of brucellosis. Therefore, we have developed a rapid vertical flow technique (RVFT) using Prussian blue nanoparticles (PBNPs) as a marker material for the detection of brucellosis antibodies. Lipopolysaccharide (LPS) was purified and used to detect brucellosis antibodies to improve the sensitivity of this technique. To enhance the sensitivity of serum antibody detection, a single multifunctional compound buffer was created using whole blood as a biological sample while retaining the advantages of typical lateral flow immunoassays. After signal amplification, standard Brucella-positive serum (containing Brucella antibody at 4000 IU mL-1) could be detected in this system even at a dilution factor of 1 × 10-2. The detection limit was 40 IU mL-1, which is ten times that before signal amplification. This RVFT displayed good specificity and no cross-reactivity. This RVFT effectively avoided the false negative phenomenon of lateral flow immunoassays, was easy to operate, had a short reaction time, has good repeatability, and could elicit results that were visible to the naked eye for 2 ~ 3min without any equipment. Since this method is very important for controlling the prevalence of brucellosis, it holds great promise for application in primary medical units and veterinary brucellosis detection.

Detection frequencies and viral load distribution of parvovirus B19 DNA in blood and plasma donations in England.

Infections with human parvovirus B19 (B19V) are transmissible by blood components and plasma-derived medicines. The European Pharmacopoeia regulates maximum levels of virus allowed in manufacturers' plasma pools. To evaluate contamination risk prior to re-introduction of UK-sourced plasma for manufacturing, we investigated viraemia frequencies of B19V in plasma samples collected from blood donors before and during COVID-enforced lockdown. Quantitative PCR for B19V DNA was used to screen pools of 96 anonymised plasma samples collected in England from 2017 (n= 29 505), 2020 (n= 3360) and 2021 (n= 43 200). Selected positive pools were resolved into individual samples. Data on donor notifications and related lookback investigations were collected from European countries by on-line survey in 2020. Screening of 76 065 donations identified 80 B19V-positive pools. While most positive samples had low viral loads (<105 IU ml-1 ), primarily from 2017 (77/29 505; 0.3%), two contained high levels of B19V DNA (1.3 × 108 and 6.3 × 106 IU ml-1 ), both likely to contaminate a final manufacturer's pool and lead to discard. The incidence of B19V infection during lockdown was reduced (1/3360 in 2020; 0/43 200 in 2021). Genomic analysis of positive pools resolved to single samples identified B19V genotype 1 in all nine samples. Seroprevalence of anti-B19V IgG antibodies was 75% (143/192). A survey of B19V screening practices in Europe demonstrated considerable variability. Two blood establishments informed infected blood donors of positive B19V results. Information on seroprevalence, incidence and viral loads of B19V viraemia is contributory the evaluation of alternative operational screening strategies for plasma testing.

Is Better Standardization of Therapeutic Antibody Quality in Emerging Diseases Epidemics Possible?

During the ongoing COVID-19 epidemic many efforts have gone into the investigation of the SARS-CoV-2–specific antibodies as possible therapeutics. Currently, conclusions cannot be drawn due to the lack of standardization in antibody assessments. Here we describe an approach of establishing antibody characterisation in emergent times which would, if followed, enable comparison of results from different studies. The key component is a reliable and reproducible assay of wild-type SARS-CoV-2 neutralisation based on a banking system of its biological components - a challenge virus, cells and an anti-SARS-CoV-2 antibody in-house standard, calibrated to the First WHO International Standard immediately upon its availability. Consequently, all collected serological data were retrospectively expressed in an internationally comparable way. The neutralising antibodies (NAbs) among convalescents ranged from 4 to 2869 IU mL-1 in a significant positive correlation to the disease severity. Their decline in convalescents was on average 1.4-fold in a one-month period. Heat-inactivation resulted in 2.3-fold decrease of NAb titres in comparison to the native sera, implying significant complement activating properties of SARS-CoV-2 specific antibodies. The monitoring of NAb titres in the sera of immunocompromised COVID-19 patients that lacked their own antibodies evidenced the successful transfusion of antibodies by the COVID-19 convalescent plasma units with NAb titres of 35 IU mL-1 or higher.

Effects of insulin on sperm cell quality in ram semen cooled at 5°C

Insulin is present in the seminal plasma and is involved in sperm activities like motility and capacitation. However, the effects of insulin on the viability of cooled ram sperm are not fully understood. Therefore, the objective of the current study was to evaluate the effect of insulin addition on ram sperm maintained at 5ºC. Sperm samples were collected from six healthy, mature Santa Inês rams. The ejaculates were divided into two aliquots with (insulin group) or without (control group) insulin (3 IU mL-1) in the semen extender, and then cooled at 5°C for 48 hours. Subsequently, the sperm cells were evaluated for motility and kinetics using computer-assisted semen analysis. The samples were evaluated for acrosomal integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and membrane functionality by the hypoosmotic swelling test. The semen analysis was performed after 24 or 48 hours of cooling. There was an increased percentage of progressive sperm motility (%), straightness (%), linearity (%) and beat caudal frequency (Hz) in the insulin group after 24 and 48 hours of cooling (p &lt; 0.05). However, insulin did not affect total sperm motility, sperm velocities (VSL, VAP and VCL) (μm seg-1), acrosomal integrity and membrane functionality during cooling (p &gt; 0.05). In conclusion, the addition of 3 IU mL-1 insulin to ram semen extender improved the quality of sperm motility after cooling.

Production of multienzyme by Bacillus aestuarii UE25 using ionic liquid pretreated sugarcane bagasse.

The utilization of sugarcane bagasse (SB) in fermentation requires pretreatment processes to render fermentable components available to microorganisms. Pretreatment by using ionic liquids (ILs) is considered promising but the high cost is an impediment in its adoption, therefore, a mixture of IL pretreated and untreated SB was utilized to obtain bacterial multienzyme under solid-state fermentation (SSF). Bacillus aestuarii UE25, a thermophilic strain was utilized for that purpose. Fermentation conditions were optimized by adopting a central composite design. The model showed a good correlation between the predicted and the experimental values for amylase, xylanase, endoglucanase, and β-glucosidase. Volumetric and specific productivity of xylanase (4580 IU ml-1 h-1 , 244.25 IU mg-1 substrate, and 50 IU mg-1 protein) were higher than the other enzymes. Changes in lignin content and reduced cellulose crystallinity due to IL pretreatment, followed by fermentation, were visualized by scanning electron microscopy, Fourier transform infrared spectroscopy, and Nuclear magnetic resonance. The strategy adopted by utilizing a mixture of IL pretreated and untreated SB under SSF proved promising to obtain high titers of different enzymes simultaneously. Since the bacterial strain used is thermophilic, therefore, the multienzyme can find its application in commercial processes which are carried out at high temperatures.