The Arctic is experiencing rapid changes in sea-ice seasonality and extent, with significant consequences for primary production. With the importance of accurate monitoring of spring phytoplankton dynamics in a changing Arctic, this study further examines the previously established critical relationship between spring phytoplankton bloom types and timing of the sea-ice retreat for broader temporal and spatial coverages, with a particular focus on the Pacific Arctic for 2003–2019. To this end, time-series of satellite-retrieved phytoplankton biomass were modeled using a parametric Gaussian function, as an effective approach to capture the development and decay of phytoplankton blooms. Our sensitivity analysis demonstrated accurate estimates of timing and presence/absence of peaks in phytoplankton biomass even with some missing values, suggesting the parametric Gaussian function is a powerful tool for capturing the development and decay of phytoplankton blooms. Based on the timing and presence/absence of a peak in phytoplankton biomass and following the classification developed by the previous exploratory work, spring bloom types are classified into three groups . Our results showed that the proportion of under-ice blooms was higher in the Chukchi Sea than in the Bering Sea. The probable under-ice blooms registered as the dominant bloom types in a wide area of the Pacific Arctic, whereas the marginal ice zone bloom was a relatively minor bloom type across the Pacific Arctic. Associated with a shift of sea-ice retreat timing toward earlier dates, we confirmed previous findings from the Chukchi Sea of recent shifts in phytoplankton bloom types from under-ice blooms to marginal ice zone blooms and demonstrated that this pattern holds for the broader Pacific Arctic sector for the time period 2003–2019. Overall, the present study provided additional evidence of the changing sea-ice retreat timing that can drive variations in phytoplankton bloom dynamics, which contributes to addressing the detection and consistent monitoring of the biophysical responses to the changing environments in the Pacific Arctic. Phytoplankton blooms in the Arctic have typically been defined as enhanced growth of phytoplankton populations that occur along the seasonally retreating ice edge, with water column stratification from sea-ice melt providing sufficient light for photosynthesis . Such maArctic annual surface air temperatures in 2014–2018 were higher than in any prior years since observational records began in 1900 . The mosa (chla) concentration, which is a proxy of phytoplankton biomass at the sea surface, Lowry et al. [a concentrations at the same time of the sea-ice retreat, indicating that there had occurred phytoplankton growth under the ice, whereas PUI blooms were defined as low chla concentrations for the first several weeks of the ice-free period because UI blooms assumed to be matured and depleted nutrients before the ice retreat. MIZ blooms were characterized as low chla concentrations at the time of sea-ice retreat and those chla concentrations increased with time to have blooms days to weeks after the ice retreat. Using a simple bloom classification approach based on the timing when the satellite-derived chla reached a bloom threshold of 2.5 mg m−3 following the retreating sea-ice edge, Lowry et al. [Based on satellite-retrieved time-series of chlorophyll-y et al.  exploredy et al.  reportedThe Pacific Arctic, specifically the region extending from the northern Bering Sea to the southern Chukchi Sea , is one a required to distinguish accurately between spring phytoplankton bloom types [a threshold [With the importance of accurate monitoring spring phytoplankton dynamics in a changing Arctic, this study examines the previously established critical relationship between the spring phytoplankton bloom types and timing of the sea-ice retreat for broader temporal and spatial coverages, with a particular focus on the Pacific Arctic for 2003–2019. To this end, time-series of satellite-retrieved phytoplankton biomass during the period of seasonal ice edge retreat were fit to an expected growth curve to identify the phase of the bloom using a parametric Gaussian function, as an effective approach to capture the development and decay of phytoplankton blooms –23. The om types . Moreovehreshold , our appThe current study is a contribution to a special issue on the Distributed Biological Observatory (DBO), specifically to highlight the potential for satellite remote sensing in supporting point and transect-based measurements in a complex environment. The DBO is a change detection array for select ecosystem variables along eight sampling transects in the Pacific Arctic . The oveRrs(λ)) at 9-km resolution at four wavelengths , obtained by the Moderate Resolution Imaging Spectroradiometer (MODIS) sensor onboard the Aqua satellite, were downloaded from NASA’s Ocean Color website (https://oceandata.sci.gsfc.nasa.gov), for the period of 2003–2019. In addition, the Advanced Microwave Scanning Radiometer (AMSR) Unified daily sea-ice concentrations gridded at a 12.5-km resolution for the period of 2003–2019 were downloaded from the National Snow and Ice Data Center website (https://nsidc.org/). AMSR Unified sea-ice concentrations were created by reprocessing the Advanced Microwave Scanning Radiometer-Earth Observing System (AMSR-E) sensor onboard the Aqua satellite during 2003–2011 and Advanced Microwave Scanning Radiometer 2 (AMSR-2) sensor onboard the Global Change Observation Mission 1st-Water (GCOM-W1) during 2013–2019. To align the spatial resolution of satellite data used in this study, daily sea-ice concentrations were resampled onto the 9-km grid using nearest-neighbor interpolation [Level-3 daily standard mapped images of remote-sensing reflectance (a (chla) concentration was computed from Rrs(λ) using the Arctic-OC3L algorithm developed and validated in the Chukchi Sea [a and b are empirically derived values of 0.3364 and −3.4388, respectively, and R is the base-10 logarithm of the maximum band ratio of Rrs(λ) among the three potential blue wavelengths  and the green wavelength .Chlorophyll-kchi Sea . The Arca over a 20-day of MIZ retreat period following the date of sea-ice retreat (DSR) were fit to an expected growth curve to identify the phase of the bloom, i.e., UI, PUI, or MIZ bloom. It is noteworthy that the 20-day of MIZ retreat period was widely used in previous studies [a was captured at a given location, phytoplankton blooms in spring were classified only as such if the pixel contained a minimum of one chla value per week and at least one chla concentrations within two days after onset of ice retreat [a time-series during the MIZ period was modeled as a Gaussian function using MATLAB lsqnonlin  on a pixel-per-pixel basis as follows:t represents the date, chla0 is the background chla, chlam is the amplitude of the chla peak observed on the date tm, and σ corresponds to the number of days chla(t) exceeded half of chlam , the chla time-series was classified into either an UI bloom or MIZ bloom. The distinction between UI and MIZ blooms is based on the model parameter tm: a chla time series was classified as an UI blooms if tm was observed before DSR (tm < DSR); in contrast if tm was equal to or after DSR (tm ≥ DSR) the bloom was classified as a MIZ bloom. In the case that the models were statistically insignificant (p ≥ 0.05), chla time-series is expressed as a constant at chla0 as follows:Then, the chlom types  based ona time-series as a single term of chla0 were classified as PUI blooms. This is based on the assumption that the pixels had already experienced a bloom before DSR, and thus the chla retrievals during the MIZ retreat period were remnants of the UI bloom [Modeled chlUI bloom .The classification was based on the assumption that pre-bloom surface nutrient concentrations are high throughout the Pacific Arctic, such that a phytoplankton bloom would be expected in waters with sufficient light availability. This assumption is reasonable because the vertical turbulent mixing of nutrient-rich Anadyr Water and Bering Shelf Water provide sufficient nutrients throughout the water column for phytoplankton growth in spring  and sucha. To ensure the robustness of our approach, we conducted a sensitivity analysis using the minimum required number  of chla concentrations during the MIZ retreat period. First, a sample chla time-series during the MIZ retreat period was generated by averaging daily chla time-series during the MIZ retreat period for all pixels classified as UI blooms over the entire study period from 2003 through 2019. Then, five chla concentrations were randomly selected from the sample chla time-series as input data, and were modeled using the a time-series during the MIZ retreat period for PUI and MIZ blooms were also generated to examine the consistency of the resulting bloom type.In the Arctic Ocean, the extensive cloud cover limits ocean-color data availability, and in turn may hamper parametric modeling of time-series variations in satellite-derived chlaMIZ denotes averaged chla during the MIZ retreat period for 2003–2019 on a pixel-by-pixel basis .Initial value, and lower and upper bounds of model parameters apply to Eqs  and 3)..3). Thesp < 0.05) for 43% of the grid cells in the study area  for UI and MIZ blooms varied substantially, suggesting this parametric modeling approach induces substantial uncertainties in estimating these bloom characteristics. In contrast, temporal variations in chla for PUI blooms were accurately fitted by the parametric approach, because this bloom type can be expressed by a single model parameter .The results of our sensitivity analysis are shown in p = 0.14; PUI bloom, p = 0.05; MIZ bloom, p = 0.75). Proportions of each bloom type in individual years are summarized in p < 0.05) decreasing and increasing trends in the proportions of UI blooms and MIZ blooms for 2003–2019 (p < 0.05) positive and negative correlations with the proportions of UI and MIZ blooms, respectively. These findings demonstrate the influence of interannual variations in DSR on bloom typology in the Pacific Arctic. Note that the mean proportion of pixels satisfying the parametric model criteria for 2003–2019 was 7.7 ± 4.1% of ocean surface in the Pacific Arctic  in the proportions of UI and MIZ blooms accompanied by the changing timing of sea-ice retreat for the period 2003–2019  and at high concentrations (>0.5 mg m−3) within a distance of ~10–20 km from the ice edge, respectively, whereas the sub-pixel contamination results in overestimation at moderate to high concentrations (>0.05 mg m−3). Therefore, the adjacency effect could lead to an underestimation of UI blooms, whereas the sub-pixel contamination could lead to an overestimation of UI blooms. By comparing the resulting proportions of three bloom types using a more conservative ice retreat threshold, Lowry et al. [a retrievals are less significant in determining spring bloom types.Sea-ice related error impacts on satellite-retrieved chly et al. . The seay et al. : the adjy et al.  demonstry et al. . AlthougThe magnitude of the zooplankton grazing on phytoplankton growth is primarily controlled by grazer biomass which was very low in spring, when zooplankton was low , 53. Inda concentration may be subject to errors due to the optical complexity of the Arctic waters [a concentrations. Therefore, calibrating/validating the performance of our approach with in situ time-series data, such as at a mooring location, would further help assess the broader validity of these satellite-based findings. It is important to reinforce that this study provided an important insight into satellite-based monitoring of phytoplankton communities in the Pacific Arctic, which can contribute to developing and managing plans and strategies for in situ observation and/or related field activities in the future.Our sensitivity analysis demonstrated a robust performance of the parametric approach in identifying spring phytoplankton bloom types in the Pacific Arctic . Howeverc waters . Moreovec waters , hamperiAlthough monitoring phytoplankton communities using satellite always contain uncertainties, the present study demonstrated robust performance of a parametric, Gaussian fitting function for the satellite time series of phytoplankton biomass in identifying spring phytoplankton bloom types . Through


This is a case report of a child who had acyanotic congenital heart disease - ventricular septal defect (VSD) and a patent ductus arteriosus (PDA) with severe pulmonary arterial hypertension. The child underwent open-heart surgery - VSD closure with PDA ligation - and six months later was re-admitted for congenital lobar emphysema of the right middle lobe. He underwent successful right middle lobectomy of the lung six months after cardiac surgery under a one-lung ventilation technique in which application of fiber-optic bronchoscope made the surgery safer and more suitable. Congenital lobar emphysema (CLE) is a rare congenital anomaly of the lower respiratory tract with an incidence of around one in 20,000 to 30,000 live births . In CLE A two-month-old male child presented with cough, tachypnea, and minimal in-drawing for a short duration. Chest X-ray revealed right upper lobe pneumonia Figure . During However, on serial follow-up post-surgery, the child continued to have a cough and occasional in-drawing which was dramatically increasing. On evaluation, the presence of infection or congestive cardiac failure was ruled out. Repeated Echo also confirmed the success of the definitive cardiac repair. On further evaluation at a later date, hyperlucency of the right middle lobe and crowding of ribs around both the right upper and lower lobes on chest X-ray (CXR) was reported. A high-resolution CT (HRCT) scan of the thorax was done which confirmed the diagnosis of congenital lobar emphysema involving the right middle lobe. After parental counseling and weighing the benefit versus risk of surgical resection of the involved lobe, the decision of surgery was made. Doing lobectomy via thoracotomy in infancy was a huge risk as per proven literature.Due to the unavailability of the double-lumen endobronchial tubes in this age group, things become difficult for the patient as well as the surgeon. A review of the literature has highlighted the use of a single-lumen endotracheal tube with gentle ventilation as an alternative to spontaneous bilateral lung ventilation. By proper selection of the endobronchial tube, hyperinflation of the affected lobe has to be prevented. A multi-disciplinary approach was taken in this case and to make induction as well as surgical resection smoother, we performed single lung ventilation using an ultrathin fiber-optic bronchoscope of 2.8 mm. We followed our same pre-planned approach of doing scopy under spontaneous breathing and light anesthesia. Finally wedging of the scope in normal bronchus which was left main bronchus in this case was achieved very smoothly. The inner diameter of the trachea was extremely difficult to negotiate traditional endobronchial blocker which was too large in diameter for this baby, so we used a 3 Fr. Fogarty catheter. Scope confirmed the position of Fogarty in the right main bronchus and we inflated the balloon just after the cut-off of the right upper lobe bronchus under direct vision given to us by fiber-optic bronchoscope Figure . The surThe child recovered fast and was extubated three hours after the surgery. Postoperatively he was asymptomatic and tachypnea and retraction had disappeared. X-ray showed good expansion of the right-sided lung with both the upper and lower lobe expanded well and filling the gap of middle lobe resection Figure . During CLE is more commonly seen in male children with the male-female ratio being 3:1 . The mosUsually, CLE is manifested in the neonatal period or early infancy with respiratory distress but can remain silent up to adolescence also, though prenatal diagnosis is also reported ,5,8. CLEDiagnosis of the condition requires a high index of suspicion. Chest X-ray can reveal the diagnosis but there are high chances that it is overlooked or misreported as collapse/consolidation due to compression of the surrounding lung . SometimCongenital lobar emphysema poses a diagnostic dilemma for management. Asymptomatic patients not in respiratory distress can be managed medically, but for those who show signs of distress, surgical resection of the diseased lung is advocated. One should prevent the ventilator-related hyper expansion of the diseased lung by maintaining low settings of pressures and volumes during ventilation at the time of surgery. As surgical mortality of 3-7% is low, as far as possible surgery should be considered for symptomatic patients instead of conservative management which accounts for 50-75% mortality [In our case the child initially presented with recurrent lower respiratory tract infections (LRTIs). During the stay, clinical suspicion led us to perform 2D Echo, which revealed a large VSD and moderate PDA with severe PAH. Our entire focus was shifted over congenital heart defect and thus post-infectious bullae rupture and pocket of pneumothorax were kept as a closer possibility rather than CLE which is rarer than large VSD in Indian scenario.We describe a very safe, suitable, and novel approach where single lung ventilation was done with 3 French Fogarty catheter aided with ultrathin 2.8 mm external diameter fiber optic bronchoscope in a high-risk young child admitted for right middle lobectomy of congenital lobar emphysema. Use of scope-guided insertion of very easily available, inexpensive tool (3 French Fogarty) to achieve single lung ventilation, rather than using very expensive and not easily available endobronchial blocker was extremely helpful to the surgeon to dissect and repair diseased emphysematous lobe without any per-operative cardiovascular compromise due to selective lobe deflation. With this approach, we insist on the role of a multidisciplinary approach in any pediatric cardio-thoracic ICU (intensive care unit) as well as an indispensable need of a dedicated pediatric cardiac intensivist in the team with modern noninvasive latest gadgets like fiber optic bronchoscope which would improve pediatric and infantile cardiac surgery repair results dramatically.


Luminescent nanomaterials have attracted recent attention as imaging and therapeutic agents for biomedical science because they provide the advantages of non-invasion, high brightness, and easy biofunctionalization. This type of nanomaterial has become a powerful tool for visualizing tissues with cellular or sub-cellular resolution and mapping molecular events, and thus shows great potential in precision medicine that seeks to tailor specific therapies to individual patients. For application in precision medicine, the material interaction within living systems should also be considered and evaluated, which includes research on the material interface and photothermal effect in the physiological environment.This Research Topic intends to present the cutting-edge of functionally developed luminescent nanomaterials with potential translational values, as well as the material interaction within living systems. The studies will contribute to the first-hand data in this field, providing a good reference for the clinical applications of the luminescent nanomaterials. This collection also encourages scientists to develop novel luminescent nanomaterials for the precise treatment of human diseases, and to explore the luminous mechanisms of these materials.Yi et al. investigated the antibacterial effect of a AIE photosensitizer, TBP-1, against group B streptococcus (GBS) and the underlying mechanism. They found that TBP-1 killed GBS in either dark or light conditions. Interestingly, light exposure further enhanced the antibacterial effect of TBP-1 by inducing damage and morphological changes of membrane of GBS. He et al. summarized the recent advances on the molecular design of AIE photosensitizers and AIE dots-based probes including tetraphenylethylene and derivatives, triarylamine and derivatives and others. They also reviewed the reported applications of AIE photosensitizer for anticancer photodynamic therapy alone or in combination with chemotherapy, photothermal therapy and radiotherapy. The author discussed the challenges of this field including targeted delivery, treatment efficiency in vivo, development of novel AIE luminogens of photodynamic therapy and uncovering new mechanism of action of AIE photosensitizer.Aggregation-induced emission (AIE) luminogens have emerged as novel phototherapeutic agent with high photostability and capacity to generate reactive oxygen species. AIE photosensitizer emits intensely at high concentrations and overcome the aggregation-induced quenching effect of common fluorescence dyes. Wu et al. utilized competitive host–guest interaction to detect cholesterol by chemical luminescence. This assay system consists of Fe3O4@SiO2 nanoparticles with the β-cyclodextrin (β-CD) decoration on their surface and ferrocene–Hemin complex that included through host–guest interaction with β-CD. Ferrocene-Hemin can be released upon the incubation with cholesterol through the competitive interaction between ferrocene–Hemin and cholesterol. Fe3O4@SiO2 nanoparticles containing cholesterol can be attracted to the bottom of assay tube by a magnet. The released ferrocene-Hemin thus catalyzes luminol/H2O2 reaction, the signal of which can be captured by a smartphone. This method has been successfully applied to quantify serum cholesterol after a simple liquid extraction process.Feng et al. developed a strategy of antibacterial therapy that combines photodynamic therapy and sliver. They constructed a self-assembled nano-carrier consisting of photosensitizer (TCPP), anti-inflammatory agent (methotrexate), Poloxamer 407 and gallic acid. Silver element was further introduced through the reduction reaction of Ag+ that catalyzed by gallic acid. The synthesized nano-carrier showed antibacterial effect against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) under the exposure of 620 nm laser.Huang et al. fabricated a cell membrane camouflaged nanoparticles to delivery DiR, a PAI contrast agent, for tumor imaging. DiR was encapsulated into poly (lactic-co-glycolic acid (PLGA) nanoparticles by emulsification and the synthesized nanoparticles were further coated with cancer cell membrane for homotypic targeting. This nanocarrier increased the circulation time of DiR in the blood and can be utilized for tumor imaging in mice.Photoacoustic imaging (PAI) has emerged as a potential non-invasive diagnosis method in clinical applications. In brief, this Research Topic presents some leading-edge researches on the evaluation of functionally developed luminescent nanomaterials, which shows great potential in the precise treatment of human diseases.


We found a very significant increase in cell engraftment in preirradiated rats. In this study, we demonstrate for the first time that preconditioning the infarcted cardiac tissue with local irradiation can substantially enhance cell engraftment.The success of cell therapy for the treatment of myocardial infarction depends on finding novel approaches that can substantially implement the engraftment of the transplanted cells. In order to enhance cell engraftment, most studies have focused on the pretreatment of transplantable cells. Here we have considered an alternative approach that involves the preconditioning of infarcted heart tissue to reduce endogenous cell activity and thus provide an advantage to our exogenous cells. This treatment is routinely used in other tissues such as bone marrow and skeletal muscle to improve cell engraftment, but it has never been taken in cardiac tissue. To avoid long-term cardiotoxicity induced by full heart irradiation we developed a rat model of a catheter-based heart irradiation system to locally impact a delimited region of the infarcted cardiac tissue. As proof of concept, we transferred ZsGreen Recent research efforts have focused on preconditioning transplantable cells to overcome this stressful environment where donor cells face the inflammatory and fibrotic tissue with low available concentrations of oxygen and glucose that complicate their survival  and cardiac troponin T (cTnT)  diluted in blocking solution . Then, tissue sections were washed in PBST (PBS containing 0.05% Tween 20) and incubated with goat anti-rabbit AlexaFluor-647  and goat anti-mouse AlexaFluor-546  secondary antibodies, diluted 1:1000 in PBS. All tissue sections were washed before being mounted in DAPI and analyzed under an automated fluorescence microscope (Zeiss Axio Imager M1).ZsGreen fluorescence signal detection and appropriate tissue preservation were assessed previously using mouse hearts . BrieflyRats were irradiated using brachytherapy. The radioactive source (Ir-192) was connected to an afterloader system (Elekta Flexitron), which places the source in the right position inside the rat for a short time. The correct positioning of the catheter implanted in the rats with regard to the target region was verified using a computed tomography (CT) scan (Siemens Sensation 16).The scanning protocol is shown in The CT images  were use+ areas were selected. For quantitative analyses, we selected the slide containing the section with the largest number of ZsGreen+ area/s, and the following previous and subsequent slides. Thus, we analyzed 9 heart sections (3 sections/slide) in each recipient rat, which represents 1.67 mm of the length of the heart  and those containing ZsGreenhe heart . ZsGreen+ areas from both groups, non-irradiated  and irradiated , together with the median of each data group were calculated. Statistical analyses were performed using GraphPad Prism software. The median of each data group is shown since data did not show a normal distribution . Statistical comparison between the two groups was performed using two-tailed, Mann-Whitney U tests. The p-value (p < 0.0001) obtained was considered to indicate a very significant difference between the two groups.ZsGreen


Gla rich protein (GRP) is a vitamin K dependent protein, shown to function as an inhibitor of pathological calcification and as an anti-inflammatory agent, with potential therapeutic use for age-related diseases such as osteoarthritis (OA) ,2. OA isNanoparticles of fluorescein-labelled chitosan/TPP with and without GRP  were prepared by ionic gelation . ResultiThe average size determined for NG was increased relatively to the NP, while flow cytometry and TEM analysis indicate the presence of GRP in NG, suggesting an effective incorporation of human recombinant GRP. Flow cytometry studies confirmed the cellular uptake of nanoparticles by macrophages. The GRP-loaded nanoparticles were able to reduce the production of TNFα in LPS-stimulated macrophages.The results confirm that chitosan/TPP nanoparticles are excellent drug delivery vehicles for GRP in macrophages and predict a wider therapeutic application in chronic inflammation-related diseases. GRP-containing nanoparticles will be further used in OA functional assays and the results will bring new knowledge on the role of GRP in the interplay between inflammation and mineralisation events associated with OA.


Bradyarrhythmia during COVID19 illness carries prognostic significance. Electrophysiological side effects of COVID19 vaccine remain largely unknown. It is imperative to report nature of cardiovascular side effects of the vaccine.An 80 years-old-man presented with complains of dizziness, trepidation and shortness of breath following his first shot of COVID-19 BBIBP-CorV (Sino-pharm). ECG on arrival showed 2:1 atrioventricular block with an underlying old left bundle branch block. The AV block changed into Mobitz type-I over the course of next 2 days and into a sinus 1:1 conduction on fourth day of presentation. However, our patient underwent permanent pacemaker implantation due to the underlying conduction tissue disease and intermittent 2:1 AV block during the hospital stay.It is likely that patients with an already diseased conduction system are at an increased risk of worsening of AV block following inoculation of the vaccine. Vaccine associated AV blocks are likely to be reversible. Presence of prior coronary artery disease and electrical abnormalities are important considerations.COVID-19 vaccine may have added side effects in subjects with known heart disease. Humoral response towards the vaccine might interfere with the conduction system of the heart and more so in patients with diseased and scarred myocardium. •Patients with underlying existing conduction tissue disease might be at an increased risk of worsening of AV block following inoculation of BBIBP-CorV vaccine.•Decision to implant permanent pacemaker should be indivisualised based on presence of prior coronary artery disease, conduction tissue abnormality, overall life expectancy and symptom correlation. BradyarThe AstraZeneca vaccine analysis report published on April 01, 2021, has reported few heart blocks, the details of which are not known . The litThe case report has been reported in line with the SCARE 2020 criteria .2An 80-year-old-man presented with complains of multiple episodes of dizziness and trepidation for 3 days and shortness of breath for one day. On arrival, patient had blood pressure of 160/100 mmHg, heart rate of 46 beats per minute (BPM), respiratory rate of 19 breaths per minute and oxygen saturation of 94% on room air. Physical examination was suggestive of a regular pulse and bi-basal chest crackles. There was no postural drop or neurological deficit. There were no signs of peripheral hypoperfusion such as cold peripheries, reduced urine output, or altered mentation. Precordial examination revealed no added sound or murmur. All peripheral pulses were palpable and equal. The patient was known hypertensive and had history of coronary artery bypass grafting (CABG) 18-years ago. Drug history included: Aspirin, Ivabradine and amlodipine . Patient had no known addictions and psychosocial history was insignificant. Patient has had no previous similar episodes. Five days before the presentation, patient had received his first shot of BBIBP-CorV (Sino-pharm).Electrocardiogram (ECG) on arrival showed 2:1 atrioventricular (AV) block with an atrial rate of approximately 75 BPM and a ventricular rate of approximately 38 BPM. The PR interval of conducted P wave was 280 msec and there was an underlying old left bundle branch block (LBBB) . LaboratEchocardiogram was done which showed an ejection fraction (EF) of 60% along with mild mitral regurgitation, moderate tricuspid regurgitation, and moderate pulmonary arterial hypertension. Owing to the hemodynamic stability, and absence of signs of hypoperfusion, temporary pacemaker (TPM) was not inserted. Patient was admitted to the Coronary Care Unit (CCU) of the hospital. Ivabradine was discontinued and patient was observed for any change in rhythm. On day three of CCU admission, patient's rhythm changed to Mobitz type-I AV block with a serial PR interval of 280 and 360 msec followed by a dropped beat . Older EPatient was further observed and mobilized cautiously with no resultant worsening of AV block. On day four of hospitalization, patient's rhythm changed to a normal sinus rhythm with 1:1 P and QRS conduction . The PR Possible differentials for an acute AV block were electrolyte imbalance, ivabradine and conduction system disease progression. Disease progression was indeed present due to an increase in first degree AV block. However, the reversibility in P to QRS conduction abnormality pointed towards an acute insult which resulted in a new onset symptomatic bradycardia. Persistence of AV block even after correction of sodium suggests that hyponatremia was unlikely to be the culprit. Our patient was on Ivabradine for last many years, however, again the acuteness of onset goes against the ivabradine being culprit. Hence, it was concluded that COVID-19 vaccination transiently worsened the AV block in our patient. Myocarditis was ruled out because of negative cardiac biomarkers and normal EF.Because of the age, symptomatic documented bradycardia, an underlying AV node (first degree AV block) and infra-HIS disease (LBBB), an expected life expectancy of more than one year, and because of the recurrence of intermittent 2:1 block during the hospital course, a decision to place a dual-chamber permanent pacemaker (PPM) was made. Patient underwent successful device placement . The pro3In a phase I/II BBIBP-CorV (Sino-pharm) vaccine trial conducted in Henan province of China, no atrioventricular block was reported . HoweverThe concept of humoral immune response and AV block is not novel. The association of maternal anti-Ro and anti-La antibodies and congenital complete heart block (CHB) is well known . Like thVaccine-associated AV block has been reported in literature after inoculation of small-pox vaccine in 2003 . In thisThe transient nature of AV block in our patient following COVID-19 vaccination can be linked to the pathophysiology of transient and various degrees of heart blocks associated with Lyme carditis. This was evident in an experiment on non-human primates injected with Borrelia burgdorferi strains, whereby an increase in IgG and IgM levels in recipients was observed. There was an accompanying increased deposition of complement-membrane attack complex . The revOur patient presented with the onset of symptoms 3 days following COVID-19 vaccination. The time scale (within first 7 days of inoculation) between vaccination and worsening of AV block points towards possible association. Additionally, the reversibility of AV block in next few days also points towards an acute insult rather than a chronic irreversible worsening of conduction system tissue. It is likely that patients with an already diseased conduction system are at an increased risk of worsening of AV block following inoculation of the vaccine.The role of steroids in COVID-19-vaccine associated AV block needs further research . It can Given the extensive use of vaccination in general population including patients with ischemic heart diseases, reporting such cases is imperative. Physicians should be cognizant of the possibility of worsening of AV block following vaccination. Watchful waiting strategy can be applied in patients with low-risk baseline ECG whereas an early pacemaker implantation can be considered in patients with high-risk baseline ECG and having an indication for PPM.4•Patients with underlying existing conduction tissue disease might be at an increased risk of worsening of AV block following inoculation of BBIBP-CorV vaccine.•Reversibility or intermittency is commonly seen with vaccine- or antibody-associated AV blocks.•The decision to implant PPM in vaccine-associated AV block should be individualized, as discussed below.•In patients with absence of an underlying coronary artery or conduction system disease, COVID-19 vaccine associated AV block might be transient and watchful strategy can be employed.•In presence of an underlying coronary artery or conduction system disease, one must be considerate of the symptom-bradycardia correlation, the degree of underlying block, patient's life expectancy and the overall benefit of device implantation.•EMB might emerge as a prognostic tool to guide decision for PPM implantation in patients with transient nature of AV blocks following a humoral response.Not commissioned, externally peer reviewed.Written informed consent was obtained from the patient.None.Pirbhat Shams: Manuscript writing, literature search and final drafting.Jamshed Ali, Sheema Saadia: literature search and drafting.Javaid Tai, Aamir Hameed Khan and Fateh Ali Tipoo: Supervision from conceptualization to editing final manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Dr. Javed Tai.None of the authors has any conflict of interests to declare.


S) Heisenberg dimers in presence of an external magnetic field. The mutual interplay between the spin size S, XXZ exchange and uniaxial single-ion anisotropy is thoroughly examined with a goal to tune the degree and thermal stability of the pairwise entanglement. It turns out that the antiferromagnetic spin- Heisenberg dimers exhibit higher degree of entanglement and higher threshold temperature in comparison with their ferromagnetic counterparts when assuming the same set of model parameters. The increasing spin magnitude S accompanied with an easy-plane uniaxial single-ion anisotropy can enhance not only the thermal stability but simultaneously the degree of entanglement. It is additionally shown that the further enhancement of a bipartite entanglement can be achieved in the mixed spin- Heisenberg dimers, involving half-odd-integer spins S. Under this condition the thermal negativity saturates at low-enough temperatures in its maximal value regardless of the magnitude of half-odd-integer spin S. The magnetic field induces consecutive discontinuous phase transitions in the mixed spin- Heisenberg dimers with The concept of negativity is adapted in order to explore the quantum and thermal entanglement of the mixed spin-(1/2, Extraordinary correlations between subsystems of a quantum-mechanical ensemble, known as entanglement, belongs to the most fascinating phenomena attracting a lot of attention during the last few decades. A huge concerment in this field of study closely relates to perspective applications of this phenomenon in a quantum computing ,2,3,4, qS) Heisenberg dimers involving the uniaxial single-ion anisotropy and magnetic field simultaneously with the special goal to verify whether the interplay between the uniaxial single-ion anisotropy, magnetic field and the spin magnitude S can enhance thermal entanglement at high enough temperatures. It should be emphasized that the uniaxial single-ion anisotropy may be relevant for several heterodinuclear complexes as for instance in magnetic compounds ·2D2O .S) Heisenberg dimer will be defined in S will be discussed in S) Heisenberg dimers will be also discussed for an arbitrary spin-S. Finally, some concluding remarks are given in The paper is organized as follows. The investigated mixed spin- Heisenberg dimers with an uniaxial single-ion anisotropy under the influence of the external magnetic field defined through the following Hamiltonian:Let us consider the mixed spin- Heisenberg dimers we will employ the concept of Peres–Horodecki [In order to study the quantum and thermal entanglement of the mixed spin- Heisenberg dimers, whereas the negativity completely vanishes  Heisenberg dimers ρ^T1/2 paNote that the density matrix S two-by-two blocks determined by basis state vectors with S) Heisenberg dimers with specific spin values Other 2envaluesλStmz∓=12envaluesλStmz∓=12g of both types of magnetic ions is set equal to two  Heisenberg dimers with The behaviour of the quantum negativity of the mixed spin- Heisenberg dimers due to the Zeeman’s splitting of energy levels and the quantum negativity of an arbitrary mixed spin- Heisenberg dimer in a state with the total spin S) Heisenberg dimer shows, in agreement with Equation  Heisenberg dimers assuming easy-plane single-ion anisotropy S) Heisenberg dimers, offers a relative simple alternative how to enhance the bipartite entanglement. The origin of qualitatively different behaviour of the negativity below and above S, but the difference S magnitude enlarges according to the formulaS, which means that the denominator in rewritten form of Equation N=4±〉 nd state  and fina)±〉 ones .S reduces the quantum as well as low-temperature thermal negativity. On the other hand, the increasing spin magnitude S enlarges the threshold temperature, which subsequently allows us to detect a subtle enhancement of the thermal entanglement at larger temperatures if the spin size S increases. The obtained results are in a perfect quantitative agreement with previous observations for the Heisenberg dimers without the uniaxial single-ion anisotropy Focusing on ropy D/J ,29,33,34S can enhance not only the threshold temperature, but it can also enhance the degree of thermal entanglement in contrast to previous knowledge. It should be emphasized that the above statement holds provided that the ensemble of integer or half-odd-integer spins S is taken into account separately. At low magnetic fields, the thermal negativity of Heisenberg dimers consisting of both half-odd-integer-spins always saturate in the maximal value S can reach the maximum value The most significant finding follows from y-axis determining the respective asymptotic values of the negativity in zero-field limit.Furthermore, in S) Heisenberg dimers exhibit very specific oscillating changes of the negativity at low and moderate temperatures (S) Heisenberg dimers with higher spin magnitude In agreement with general expectation the increasing temperature reduces the bipartite entanglement with a significant drop of the negativity emergent in a proximity of all magnetic-field-induced phase transitions associated with crossing of energy levels. Around each level-crossing field the interplay between thermal and quantum fluctuations is the most pronounced and the negativity shows a marked local maximum located between two neighbouring level-crossing fields. In a consequence of that, the mixed spin- Heisenberg dimers are more persistent against rising temperature and magnetic field than their ferromagnetic counterparts  Heisenberg dimers. In particular, it has been verified that the concurrent interplay of the uniaxial single-ion anisotropy and the external magnetic field basically influences bipartite entanglement of the mixed-spin Heisenberg dimers. To quantify the degree of bipartite entanglement we have derived the exact analytical expression for the negativity in terms of Peres-Horodecki criterion [S) Heisenberg dimers as a necessarily prerequisite for further entanglement analysis. Two different scenarios were observed for both the antiferromagnetic  Heisenberg dimers with an arbitrary spin-S magnitude for the particular value of the uniaxial single-ion anisotropy S) Heisenberg dimers with both half-odd-integer spin constituents.As a direct consequence of different effect of easy-axis and easy-plane uniaxial single-ion anisotropy one detects very different influence of increasing spin magnitude Equation . In cont Formula , the enhS enlarges the threshold temperature, but unfortunately reduces the degree of thermal entanglement. However, a completely novel and unexpected behaviour has been observed for the easy-plane single-ion anisotropy S may simultaneously enlarge the threshold temperature as well as the degree of thermal entanglement if one compares solely integer or half-odd-integer spin-S cases. It has been evidenced that except the particular case with S) Heisenberg dimers with both half-odd-integer spins generally achieve higher degree of entanglement, which makes them more attractive for a practical utilization. Last but not least, a fascinating oscillating changes of the negativity were observed upon the variation of the external magnetic field at low and moderate temperatures. The unconventional oscillating behaviour of the negativity originates from existence of consecutive field-driven phase transitions emergent at level-crossing fields, at which the negativity rapidly falls down before it produces significant local maxima localized in between two level-crossing fields. It is noteworthy that the local maxima of the negativity of the mixed spin- Heisenberg dimers with integer and half-odd-integer spin S are not equal due to existence of different ground states in the parameter region of the easy-plane single-ion anisotropy The comprehensive analysis of a thermal entanglement above a unique ground state


Finally, it provides a future perspective for COVID-19 vaccine analytical tools and attempts to predict how the field might evolve over the next 5–10 years.Vaccines are key in charting a path out of the COVID-19 pandemic. However, development of new vaccines is highly dependent on availability of analytical methods for their design and evaluation. This paper highlights the challenges presented in having to rapidly develop vaccine analytical tools during an ongoing pandemic, including the need to address progressive virus mutation and adaptation which can render initial assays unreliable or redundant. It also discusses the potential of new computational modeling techniques to model and analyze key viral proteins and their attributes to assist vaccine production and assay design. It then reviews the current range of analytical tools available for COVID-19 vaccine application, ranging from  When a novel pathogen, such as SARS-CoV-2 emerges, researchers have to start from scratch and rapidly develop new assays, analytical standards and controls in a race against time. Increasing globalization and encroachment of humans on animal habitats means pandemics may become more frequent in the future [COVID-19 is a life-threatening disease that emerged in late 2019 that is caused by infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Since its initial detection, SARS-CoV-2 has infected over 200 million people globally and caused almost 5 million deaths [e future . For posIn addition to trying to better understand the novel SARS-CoV-2 virus, vaccine developers also need to contend with designing or adapting analytical tools to support the evaluation of the new vaccine technologies. The COVID-19 pandemic is unique in that a number of new vaccine platforms, namely viral vectors, mRNA- and DNA-based vaccines, have been deployed to try and counter the virus . While sin silico modeling that have proved invaluable in their vaccine development efforts.This special report first looks at the development process for new analytical tools in the context of an ongoing pandemic in which the disease mechanisms remains largely unknown and the virus presents as a ‘moving target’. It then describes the current state of play of analytical tools been utilized for COVID-19 vaccine development, ranging from optimization of vaccine design to manufacturing and quality assurance, immunogenicity and protection assessment in animals and human trials. The article provides hints, tips and in-house protocols developed by the authors during development of their own SARS-CoV-2 vaccine (Covax-19) and provides insights into emerging analytical tools including bioinformatics and in vitro assay or an in vivo preclinical model  and sequencing can be used as critical quality attributes to quickly confirm the identity of mRNA, DNA or viral vector vaccines . These tAt a proteomic level, there are a number of approaches that can be employed to validate protein-based vaccines, and to confirm the correct translation of mRNA, DNA and viral vectors into protein by host cells. Protein separation using gel electrophoresis followed by staining or western blots can confirm semi-quantitatively protein size and purity. A benefit of this technique is that it is relatively simple procedure with required equipment available in most laboratories. A limitation of western blots is that they require that an antibody is already available against the vaccine protein . More co2 production or visual changes [For vaccines produced using cell-based expression systems, there is the potential for contamination, such as host cell proteins (HCP), endotoxins and adventitious viruses. As regulatory requirements differ between regions, researchers should consult guidelines and standards released by the relevant regulatory agency such as the US FDA or EMA. Bacterial and fungal contamination can be detected using sterility tests. These tests are routinely performed in all biological manufacturing facilities and generally involve inoculating sterile media with the vaccine sample and the monitoring for 14–28 days for growth via CO changes . The ass changes  or via a changes .New classes of vaccines such as mRNA and DNA are typically chemically synthesized, however several reagents/inputs  are still produced using microbial fermentation and therefore several of the above-mentioned assays are still applicable. Contaminants specific to mRNA vaccines sythensis include oligoribonucleotide impurities as a result of abortive initiation events by RNA polymerases or double-stranded RNA generated by self-complementary 3′ extension, all of which can trigger inflammation . Traditiin vitro based assays to measure cytotoxicity, genotoxicity and carcinogenicity in which cell viability, mutations and chromosomal damage is monitored in cells following administration of vaccine components are progressively replacing in vivo toxicity testing assays [Ensuring safety is the number one priority in vaccine design. A simple test to evaluate the toxicity of biological products is the general safety test , which involves administering a defined amount of vaccine to an animal and evaluating subsequent toxicity via body weight loss, clinical scoring, temperature and/or survival . Mice arg assays .One early concern for SARS-CoV-2 vaccines was the potential for vaccine-enhanced disease as this had been reported for previous SARS ,39 and Min silico modeling can itself be used as an analytical tool to screen vaccine candidates. 3D structural models of the vaccine immunogen can be used to evaluate the impact of sequence modifications, such as removal or mutation of an enzyme cleavage site or the introduction of stabilizing mutations or the removal of the transmembrane/cytoplasmic domain to create a soluble secreted protein for easy expression and purification. Once the residue changes are incorporated into the spike protein sequence, molecular dynamic simulations (MDSs) can be run to characterize the likely impact of such changes on protein behavior such as whether the modified antigen will form a stable trimer structure similar to the native SARS-CoV-2 spike protein. This computational approach allowed the successful development of our successful vaccine candidate despite our having no access to actual SARS-CoV-2 virus to study [16 synthetic oligonucleotides in silico and then a second machine learning program named ‘Search Algorithms for Ligands’ (SAM) was used to select an optimal TLR9 agonist oligonucleotide to include as an adjuvant in our COVID-19 vaccine [In addition to being used to aid in the development of analytical tools, to study . Computato study . The tra vaccine . While tet al. [in vivo by incorporating a reporter  into the COVID-19 mRNA vaccine. In combination with follow-up histopathology, this approach can provide a picture of mRNA biodistribution and safety. A limitation of this method is that it requires animal protocol approval, is time consuming, uses specialized imaging equipment and if the reporter is fluorescence-based there may be issues with high auto fluorescent background from host tissue, whereas bioluminescent reporters can vary based on substrate kinetics and tissue uptake [The COVID-19 pandemic is unique in the number of different vaccine platforms that have been deployed against the virus. While some assays established for traditional vaccines may still be applicable to the new types of vaccines such as mRNA or DNA, others are not, requiring new vaccine-platform specific assays and benchmarks to be developed  ,52 and vn silico . Softwarn silico  and Allen silico . For virThe two main types of vaccine-induced immune responses are humoral (antibody) and cellular (B and T cell) immunity. Immune assays are required to establish an immune correlate of vaccine protection, that could then be used to guide subsequent vaccine development. A reliable immune correlate of vaccine protection then allows easy comparisons of different candidate vaccines and simplifies future vaccine approvals by avoiding the need to undertake large and expensive Phase III outcome trials. As yet, although there is some correlation seen between serum neutralizing antibody and protection, no immune correlate of protection for SARS-CoV-2 has been agreed , making Antibody responses to a vaccine are measured mainly via sera collected from venous blood. Unlike cell-based assays, sera can be easily inactivated to allow post infection samples to be safely removed from facilities where virus challenges are undertaken. It is important to run both positive and negative (naive) sera controls in each antibody assay. Examples of available positive sera controls for SARS-CoV-2 antibody assays include the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin, human (NIBSC code: 20/136) and Anti-SARS-CoV-Verification Panel for Serology Assays (NIBSC code: 20/B770). Given the global prevalence of SARS-CoV-2 infection and unprecedented vaccine rollout campaign  and the levels and ratios of these antibodies can provide information on the type of immune response induced by the vaccine. For example, cytokines produced by T helper 1 (Th1) cells and T helper 2 (Th2) cells preferentially bias production of IgG2 and IgG1 antibody subclasses, respectively . A varieSupplementary Table 1i. In brief, HeLa cells were transduced with a lentiviral vector containing the SARS-CoV-2 spike gene and this then generated stable spike-expressing cell clones (HeLa-Sp). Surface expression of spike protein on the HeLa cells was confirmed by flow cytometry and immunofluorescence staining. An initial cell fixation step was later omitted because it caused a high background. The unfixed CELISA had significantly lower non-specific background when used with human sera compared with the standard spike ELISA  assay. A full protocol for the CELISA is available in ke ELISA . VariatiA major limitation of traditional ELISA assays are they require high sample volumes and only measure one analyte at a time. Other multiplexed assay platforms including bead-based immunoassays and protein microarrays can be used for detection of antibodies and other analytes in a single sample. For example, the LabScreen COVID PLUS (Lambda) assay measures antibody responses to different fragments of SARS-CoV-2 including full spike extracellular domain, S1, RBD, S2 and nucleocapsid (NP) protein)  as well in vitro method to evaluate potential immune protection, although for some viruses such antibody mediated neutralization titers have minimal correlation with actual protection. This is likely because for many viruses T-cell immunity may be at least, if not more, important to virus protection. Hence while neutralization assays provide useful data, they are rarely the sole arbiter of whether a vaccine will be effective or not. A further problem is the difficulty in standardizing such assays as they rely on live cell substrates and these and their culture conditions may vary widely from lab to lab resulting in assay differences. Typically early in a pandemic a panel of convalescent sera is used as a benchmark with the size of the vaccine-induced antibody neutralization responses and allows for comparison to those seen in other laboratories.Neutralization assays are an Supplementary Table 1ii and [The two main types of neutralization assay include wild-type (WT) SARS-CoV-2 and pseudotyped viruses. WT viruses better mimic natural infection, however use of live SARS-CoV-2 viruses requires BSL3 facilities with strict biosafety control, which is not available to many research institutes. In addition, WT viruses can be restricted by local availability of the specific virus isolates needed for the study due to customs importation restrictions. There is also the need to successfully culture and maintain the virus stock with the risk that mutations may occur during cell culture. Pseudotype viruses are replication-deficient viruses made to express component of the target virus, in this case the SARS-CoV-2 spike protein, and these represent a potential alternative to WT virus. The major benefit of pseudotype virus assays is that they only require BSL2 lab conditions, and so can be quick to set up in most laboratories and are flexible to adapt to emerging virus mutants or variants. Our lab developed a lentivirus pseudotype virus assay with fluorescent protein for flow cytometry or imaging-based detection as well as a luciferase-based system to allow a luminescent plate reader detection method , which Recently, a number of surrogate neutralization assays have been developed to measure antibodies inhibiting SARS-CoV-2 cellular attachment . The assCapillary electrophoresis (CE) is another example of an analytical tool that can be used to assess the interactions between viruses and subviral particles  with antibodies and receptors . Viruses3H-thymidine incorporation assay to carboxyfluorescein succinimidyl ester (CFSE) dilution assay and surrogate proliferation cell markers; (2) cytokine production ; (3) assessing the frequency and phenotype of B- and T-cell populations by flow cytometry; and (4) B- and T-cell receptor  repertoire sequencing. Each method has its own advantages and disadvantages and there remains a high degree of variability between laboratories in both technical approaches and interpretation. A summary of the various analytical assays for measuring vaccine-induced cellular responses is provided in The primary inputs for analyzing antigenic-specific cellular immune memory responses are splenocytes in animals and peripheral blood mononuclear cells (PBMCs) in humans. Commonly used cellular assays measure antigen-specific B- and T-cell responses by (1) assessing antigen-stimulated immune cell proliferation, ranging from traditional +/CD8+) following stimulation with antigen-specific stimulation  in cell culture setting at different time points can be used to measure the level of T-cell memory and critical for antiviral recall responses [3H-thymidine [3H-thymidine assay is more labor intensive, requires specialized equipment and safety concerns relating to the handling and disposal of radioisotopes. An alternative approach is to assess proliferation via the dilution of cell permeant dyes, such as CFSE, cell trace violet (CTV), or violet proliferation dye 450 (VPD-450). The tracking of lymphocyte division using CFSE is a routine procedure in many laboratories. Prior to antigen stimulation, cells are stained with the cell permeant dyes and as the cells proliferate the intensity of the dye in daughter cells as measured by flow cytometry is halved with each cell division [The proliferation of PBMCs and T-cell subsets , enzyme-linked immune absorbent spot (ELISPOT) and intracellular cytokine staining (ICS) assay. A conventional method for measuring cytokines released by cells is via ELISA, however this technique is very low throughput and requires large sample volume. CBA assays use spectrally distinct beads coated with capture antibodies which allows for the simultaneous measurement of up to 30 secreted analytes from small volumes of biological samples such as supernatant, serum and cell lysate . The conELISPOT assays allow for the quantitation of the frequency of specific cytokine secreting cells . In brietrans-Golgi function [+ T-cell responses in individuals with COVID-19 [+ and CD4+ T-cell responses, respectively, by ICS following in vitro stimulation with SARS-CoV-2 overlapping peptides. A limitation of the assay is low sensitivity when frequency of antigen-specific T cells is low and the protocols require that cells are fixed which may interfere with analysis of surface markers.ICS is single-cell based FACS assay that can be used to simultaneously detect both cytokine production and the cell phenotype. Instead of detecting the levels of secreted cytokines, stimulated cells are treated with a selective protein secretion inhibitor, Brefeldin A or monensin, that causes the responding cells to retain cytokines rather than secreting them ,95. Breffunction . Monensifunction ,98. TherCOVID-19 ,100. Up + T cells [Quantifying the number of antigen-specific, MHC-restricted, T cells is important in assessing the cellular immune response generated by potential COVID-19 vaccine candidate. One of the limitations of stimulation-based functional assays, such as proliferation, ELISPOT assays and ICS, is that they only detect functional antigen-specific T cells as determined by the scope and measurement parameters of the assay(s), and therefore can underestimate total antigen-specific T cells. Tetramers can be used to capture the frequency of antigen-specific T cells regardless of functionality, overcoming the limitations of existing stimulation-based functional assays . Tetrame T cells . One of  T cells .+ and CD8+ T cells following immunization or re-stimulation in vitro. Markers expressed on T cells early after stimulation include CD69 [+ and OX40+ for CD4+ T cells and CD69+ and CD137+ for CD8+ T cells) have been used in a number of SARS-CoV-2 studies to measure T-cell memory to SARS-CoV-2 for up to 8 months after infection [T-cell responses are characterized by activation, clonal expansion/proliferation and differentiation into effector cells to mediate specific functions including memory cell formation . Fluoresude CD69 , OX40  [Antigen-specific B- and T-cell responses are governed by the specificity and selectivity of their respective BCRs or TCRs. BCR and TCR repertoires generated in an immune response can described as (i) skewed, (ii) immunodominant’, (iii) restricted or (iv) limited . Analyzi18) . The ant18) . CDR3 al18) ,125. Hig18) ,127. The18) .in vitro assays can provide useful information on vaccine antigen structure and stability, the best method to determine whether a vaccine is effective is an animal challenge model. Important considerations include the animal species, sex and age and what assays to use to assess protection. Most important is what virus will be used in the challenge and whether it needs to be adapted to ensure it is infectious in the relevant animal species. There are a number of different animal models available to evaluate SARS-CoV-2 infection and each provides different information. Types of models include (i) direct virus challenge studies, (ii) indirect passive immunization studies and (iii) virus transmission models. In direct challenge studies an animal is immunized and then challenged with SARS-CoV-2 virus to assess vaccine protection. Passive immunization studies involve the transfer of isolated antibodies from immunized animal to a naive animal that is subsequently challenged to determine whether the transferred antibodies can confer protection. The benefit of passive immunization is that you can assess the contribution of humoral immunity only in protection. In addition, antibodies can be isolated from clinical trials and transferred to animals to assess whether vaccine-induced human antibodies can confer protection against SARS-CoV-2 infection in vivo outside the patient in a controlled experimental setting. Transmission models assess whether the virus infection is transmitted from infected animals to non-infected animals and hence whether vaccination of one animal can prevent it spreading the disease to a second non-vaccinated animal.Pandemic vaccine development requires suitable animal models in which to evaluate vaccine efficacy against infection. Although The three main methods for assessing protection in animal models are weight-loss, sickness scores and histopathology analysis of tissues of interests . For histopathology analysis, tissue are fixed, sectioned and stained, namely with hematoxylin and eosin (H&E), to reveal tissue morphology. SARS-CoV-2 pathology shares similar characteristics as SARS and MERS infection . FeatureAdditional important consideration to take into account in challenge studies is the strain and dose of SARS-CoV-2 virus together with the species, age, weight and co-morbidities of the animals being challenged ,134. FurAn ideal animal model should have a similar physiology and closely replicate disease mechanisms/pathology in humans. It is also important that the receptors/pathways are similar in the animal species in order to ensure the animal results replicate the vaccine immune response and virus protection in humans. Due to homology, shared cell entry mechanisms and similar disease pathology of SARS to SARS-CoV-2, several of the previously established SARS animal models in literature were able to be rapidly adapted for SARS-CoV-2 challenges. Animal models established for SARS-CoV-2 include transgenic ACE2 mice, hamsters, ferrets and non-human primates (NHPs) . Mice arTissue for SARS-CoV-2 virus quantification can come in many formats including blood sera, tissue specimens and mucosal fluids. The sensitivities and detection rates for SARS-CoV-2 can vary depending on location and timing of specimen collection . Moreovein situ tissue staining [In situ staining using immunohistochemistry (IHC) or in situ hybridization (ISH) is another way to quantify virus infection [Virus quantification can be performed using plaque-based assays, tissue culture infectious dose (TCID50) assays , PCR assstaining . Plaque staining . Both thstaining . In situnfection . IHC invnfection . These tnfection .in vivo protection models. Other critical factors in rapid deployment of analytical tools during pandemics are open-source protocols, liberal sharing of appropriate controls/standards to allow benchmarking of results between laboratories, and rapid dissemination and publication of ongoing research into biology and disease mechanisms of the pandemic virus to ensure all analytical tools and vaccine designs remain accurate and relevant.A vaccine is only as good as the analytical tools available to evaluate it. In the event of a pandemic, such as COVID-19, there is added pressure to rapidly develop analytical tools to help bring safe and effective vaccine candidates to market in a timely manner. In this special report, we have detailed the analytical tool development process in the context of a pandemic vaccine and the common pitfalls faced by vaccine researchers and developers. Emerging technologies, such as computational modelling and bioinformatics, are helping overcome these challenges and changing the way we design and validate analytical methods and assess the vaccines candidates themselves. In this Special Report we have described the current state of analytical methods relevant to COVID-19 vaccine ranging from antigen characterization to COVID-19 is not the first global pandemic in modern history and will not be the last. Increased globalization and urbanization/human-animal contact mean that the frequency and severity of future pandemics are likely to increase . VaccineModular design: Given the increase in novel viruses and variants it is important to design modular analytical tools that are adaptable and can be readily validated so they can be rapidly deployed.Multiplex assay formats: An individual analytical assay can provide single output/measurements; however, by integrating the results and connecting datasets from different assays enable researchers to identify new trends, biomarkers, correlates of protection and vaccine antigen targetsin silico modeling, in speeding up the design and validation of vaccine antigen and analytical assays to keep pace with our changing world. Other emerging technologies such as next generation bulk and single cell RNA-sequencing can be used to broadly characterize the entire expression profile of immune cells, this approach is particular important in the context of a novel virus wherein correlates of protection are unknown and/or when new adjuvants or vaccine technologies are being employed and their mode of action has yet to be fully characterized. However a limitation of these technology platforms is the significant amount of technical knowledge and infrastructure required to run analyses. Training in these new areas of research, even starting at an undergraduate curriculum level, and increasing the availability and access to computing platforms and other specialized equipment will help with the uptake of these technologies.Integration of emerging technologies: Throughout the paper we highlight the benefit of incorporating new technology platforms, such as bioinformatics and Key analytical trends that are likely to influence the pandemic vaccine field over the next 5–10 years include:Key in pandemic vaccine analytical tool development is to tightly frame the research question that needs to be addressed, identify key inputs and design a reproducible protocol with appropriate controls to allow assessment of assay sensitivity, specificity, accuracy, reproducibility and interference.Developing analytical tools is not a fixed process but should be ongoing with the method being continually checked/validated and updated if necessary.In silico modeling tools can help speed up pandemic vaccine development by enabling researchers to rapidly design and evaluate vaccine candidates.Robust characterization of the antigen and continued quality assurance measures are necessary as the composition of the final vaccine product can influence its immunogenicity and protection.Newer functional antibody assays such as pseudotype and surrogate neutralization assays help overcome limitations of conventional virus neutralization assays.Analytical tools for use in development of pandemic countermeasures should ideally be adaptable to respond to changing circumstances including rapidly mutating viruses.Early pandemic vaccine development is hindered by lack of access to appropriate immune sera and antigen standards and creation and distribution of such standards should be an immediate priority in event of any outbreak.Click here for additional data file.


Four-dimensional flow CMR allows for a comprehensive assessment of the blood flow kinetic energy of the ventricles of the heart. In comparison to standard two-dimensional image acquisition, 4D flow CMR is felt to offer superior reproducibility, which is important when repeated examinations may be required. The objective was to evaluate the inter-observer and intra-observer reproducibility of blood flow kinetic energy assessment using 4D flow of the left ventricle in 20 healthy volunteers across two centres in the United Kingdom and the Netherlands.This dataset contains 4D flow CMR blood flow kinetic energy data for 20 healthy volunteers with no known cardiovascular disease. Presented is kinetic energy data for the entire cardiac cycle , the systolic and diastolic components, in addition to blood flow kinetic energy for both early and late diastolic filling. This data is available for reuse and would be valuable in supporting other research, such as allowing for larger sample sizes with more statistical power for further analysis of these variables. Quantitative cardiovascular magnetic resonance (CMR) imaging can provide a wealth of information to distinguish health from disease . Four-diReproducibility in cardiac MRI research is a pervasive issue across the field, which impacts the translational pathway . Given tThe dataset includes the LV blood flow KE parameters of twenty healthy adult volunteers void of any known cardiovascular disease, collected from two sites in the United Kingdom (Leeds) and the Netherlands (Leiden). CMR was performed on a dedicated cardiovascular 1.5 Tesla Phillips Ingenia system equipped with a 28-channel coil and Phillips dStream digital broadband MR architecture technology. The data was acquired following analysis of CMR images using dedicated 4D flow research software . After acquiring images, advanced 4D flow CMR techniques were applied to determine the kinetic energy (KE) for each phase of the cardiac cycle, following which all KE parameters were normalized to the LV end-diastolic volume, and reported in standard units (μJ/ml).The dataset is composed of two parts. First, inter-observer reproducibility. This refers to two separate operators analysing the twenty cases independent of each other. Second, intra-observer reproducibility. This refers to the same operator analysing the same ten cases on two separate occasions, with a three-month interval between. The data presented is the endpoint of a range of software-delivered processing of the images, which is beyond the scope of this report. Further detail on the 4D LV KE mapping can be found within our primary publication on this topic [For each case within the two reproducibility analyses, we include data for: global LV KE (an average KE of the LV flow for the complete cardiac cycle), systolic KE (the average KE of the LV flow during systole), diastolic KE (the average KE of the LV flow during diastole), peak E-wave KE (the peak KE of the LV flow during early diastolic filling), and peak A-wave KE (the peak KE of the LV flow during late diastolic filling)..The temporal resolution of 4D flow is 40 ms (ms), which may impact the precision of KE assessments. This is an issue characteristic of 4D flow analyses, rather than a limitation unique to our dataset. 4D flow was acquired during free-breathing, for which issues relating to heart rate may have impacted on the time-varying flow characteristics, which could not be corrected for. There are no other immediate limitations of this dataset


Tuberculosis (TB) and COVID-19 pandemics are both diseases of public health threat globally. Both diseases are caused by pathogens that infect mainly the respiratory system, and are involved in airborne transmission; they also share some clinical signs and symptoms. We, therefore, took advantage of collected sputum samples at the early stage of COVID-19 outbreak in Ghana to conduct differential diagnoses of long-standing endemic respiratory illness, particularly tuberculosis.Mycobacterium tuberculosis complex. Positive pools were then decoupled and analyzed individually. Details of positive TB samples were forwarded to the NTP for appropriate case management.Sputum samples collected through the enhanced national surveys from suspected COVID-19 patients and contact tracing cases were analyzed for TB. The sputum samples were processed using Cepheid’s GeneXpert MTB/RIF assay in pools of 4 samples to determine the presence of Mycobacterium tuberculosis in both suspected COVID-19 cases  and their contacts . A total of 111 (14.3%) were diagnosed with SARS CoV-2 infection and six (0.8%) out of the 774 individuals tested positive for pulmonary tuberculosis: five (83.3%) males and one female (16.7%). Drug susceptibility analysis identified 1 (16.7%) rifampicin-resistant tuberculosis case. Out of the six TB positive cases, 2 (33.3%) tested positive for COVID-19 indicating a coinfection. Stratifying by demography, three out of the six (50%) were from the Ayawaso West District. All positive cases received appropriate treatment at the respective sub-district according to the national guidelines.Seven-hundred and seventy-four sputum samples were analyzed for Our findings highlight the need for differential diagnosis among COVID-19 suspected cases and regular active TB surveillance in TB endemic settings. The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory coronavirus 2 (SARS CoV-2) was first reported in the Wuhan suburb of Hubei province in China in early December 2019 and has since spread to all regions of the world. It was officially declared a pandemic by the World Health Organization (WHO) in March 2020 . HoweverMycobacterium tuberculosis complex (MTBC) is a disease of antiquity but still remains a global health emergency  samples were received for primary diagnosis of TB compared to ninety-four [94] samples being received in the months of January and February; 2 months period. The low numbers recorded can be attributed to the reduced attention to TB as most of the health workers were primarily focused on combating COVID-19 to the neglect of other important infectious diseases . A model months wThe National Tuberculosis Control Programme (NTP) is adopting strategies to improve access to TB diagnosis and care with strategies such as early screening tools at OPDs, and ready access to TB treatment for home-based TB care. However, more needs to be done in the area of community education and engagement campaigns to improve early case detection and compliance to therapy to reduce negative outcomes of TB disease in Ghana. This pilot study also provided valuable information to the NTP and the health service on the need for differential diagnosis as well as strengthening surveillance of long-standing endemic infectious diseases amid the pandemic. More studies will however need to be conducted to understand better if latent TB infection progressed to active TB with COVID-19 infection or a relapse of a previous TB infection upon COVID-19 infection and the impact of co-infection on treatment outcome.