Tef [Eragrostis tef (Zucc.) Trotter], an allotetraploid cereal that is a staple food to over 60 million people in the Horn of Africa, has a high nutritional content and is resistant to many biotic and abiotic stresses such as waterlogging and drought. Three tef genotypes, Alba, Tsedey, and Quncho, were subjected to waterlogging conditions and their growth, physiology, and change in transcript expression were measured with the goal of identifying targets for breeding cultivars with improved waterlogging tolerance. Root and shoot growth and dry weight were observed over 22 days. Stomatal conductance and chlorophyll and carotenoid contents were quantified. Microscopy was used to monitor changes in the stem cross sections. Illumina RNA sequencing was used to obtain the expression profiles of tef under flooding and control conditions and was verified using qPCR. Results indicated differences in growth between the three genotypes. Waterlogged Tsedey plants grew higher and had more root biomass than normally watered Tsedey plants. Quncho and Alba genotypes were more susceptible to the excess moisture stress. The effects of these changes were observed on the plant physiology. Among the three tested tef genotypes, Tsedey formed more aerenchyma than Alba and had accelerated growth under waterlogging. Tsedey and Quncho had constitutive aerenchyma. Genes affecting carbohydrate metabolism, cell growth, response to reactive oxygen species, transport, signaling, and stress responses were found to change under excess moisture stress. In general, these results show the presence of substantial anatomical and physiological differences among tef genotypes when waterlogged during the early growth stage.

The Russian dandelion Taraxacum koksaghyz produces high-value isoprenoids such as pentacyclic triterpenes and natural rubber in the latex of specialized cells known as laticifers. Squalene synthase (SQS) and squalene epoxidase (SQE) catalyze key steps in the biosynthesis of cyclic terpenoids, but neither enzyme has yet been characterized in T. koksaghyz. Genomic analysis revealed the presence of two genes (TkSQS1 and TkSQS2) encoding isoforms of SQS, and four genes (TkSQE1-4) encoding isoforms of SQE. Spatial expression analysis in different T. koksaghyz tissues confirmed that TkSQS1 and TkSQE1 are the latex-predominant isoforms, with highly similar mRNA expression profiles. The TkSQS1 and TkSQE1 proteins colocalized in the endoplasmic reticulum membrane and their enzymatic functions were confirmed by in vitro activity assays and yeast complementation studies, respectively. The functions of TkSQS1 and TkSQE1 were further characterized in the latex of T. koksaghyz plants with depleted TkSQS1 or TkSQE1 mRNA levels, produced by RNA interference. Comprehensive expression analysis revealed the coregulation of TkSQS1 and TkSQE1, along with a downstream gene in the triterpene biosynthesis pathway encoding the oxidosqualene cyclase TkOSC1. This indicates that the coregulation of TkSQS1, TkSQE1, and TkOSC1 could be used to optimize the flux toward specific terpenoids during development.

Plants growing in high densities experience a reduced red (R) to far-red (FR) light ratio and shade-intolerant species respond with accelerated elongation growth to reach the top of the canopy: the shade avoidance syndrome (SAS). FR-enriched light inactivates phytochrome photoreceptors, which results in subsequent action of several plant hormones regulating growth. SAS is adaptive for shade-intolerant plants, but is suppressed in shade-tolerant plant species. Inspired by a previously published transcriptome analysis, we use two species of the genus Geranium here to study the involvement of auxin, brassinosteroids (BRs), and gibberellins (GAs) in supplemental FR-induced elongation growth. G. pyrenaicum, a shade-avoiding species, strongly induces auxin and gibberellin levels, but not BR, in elongating petioles. We show that, in this species, FR light perception, hormone synthesis, and growth are local and restricted to the petiole, and not the leaf lamina. Using chemical hormone inhibitors, we confirm the essential role of auxin and GAs in supplemental FR-induced elongation growth. Shade-tolerant G. robertianum does not display the change in hormone levels upon FR light enrichment, resulting in the lack of a shade avoidance response.

Trehalose 6-phosphate (Tre6P), a sucrose signaling metabolite, inhibits transitory starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves and potentially links starch turnover to leaf sucrose status and demand from sink organs (Plant Physiology, 163, 2013, 1142). To investigate this relationship further, we compared diel patterns of starch turnover in ethanol-inducible Tre6P synthase (iTPS) lines, which have high Tre6P and low sucrose after induction, with those in sweet11;12 sucrose export mutants, which accumulate sucrose in their leaves and were predicted to have high Tre6P. Short-term changes in irradiance were used to investigate whether the strength of inhibition by Tre6P depends on starch levels. sweet11;12 mutants had twofold higher levels of Tre6P and restricted starch mobilization. The relationship between Tre6P and starch mobilization was recapitulated in iTPS lines, pointing to a dominant role for Tre6P in feedback regulation of starch mobilization. Tre6P restricted mobilization across a wide range of conditions. However, there was no correlation between the level of Tre6P and the absolute rate of starch mobilization. Rather, Tre6P depressed the rate of mobilization below that required to exhaust starch at dawn, leading to incomplete use of starch. It is discussed how Tre6P interacts with the clock to set the rate of starch mobilization.

- The purpose of the study was to assess the level of serum malondialdehyde (MDA) concentration and its association with the stage and histopathologic sizes of colorectal cancer (CRC). One hundred and two patients having undergone surgical treatment of CRC between January 2014 and December 2015 were included in this cross-sectional study. The patients were divided into four groups (stage I-IV) according to the TNM classification. Control group included 30 subjects with no signs of malignancy and inflammatory diseases. In each patient, preoperative blood samples were obtained for determination of MDA concentration by ELISA immunoassay. Serum levels of MDA were progressively increased in patients with CRC, reaching the highest value in the fourth stage of CRC. Serum concentrations of MDA were significantly higher in pT4 group as compared with pT3 and pT2 groups of CRC patients (p<0.01). Significantly higher levels of MDA were found in the N1 and N2 groups of CRC patients as compared with N0 group, as well as in patients with metastatic disease as compared with those without metastasis (p<0.001). In conclusion, the progression of CRC is associated with a significant increase in serum MDA levels.

- According to present findings, the impact of particular arterial pressure components on the occurrence of left ventricular hypertrophy (LVH) differs. We sought to determine which individual component of arterial pressure has the greatest impact on the LVH geometric pattern/degree. The study included 192 patients (87 men), aged 43-80 (median 68) years with hypertension and LVH. Patients were classified into three groups according to type of hypertrophy (concentric, eccentric and asymmetric) and into three subgroups according to the degree of hypertrophy (mild, moderate and severe). All patients had their blood pressure measured, and they underwent electrocardiography and echocardiography. Antihypertensive drugs and the duration of previous treatments were taken into consideration. Pulse pressure was significantly higher in patients with concentric LVH than in those with eccentric and asymmetric LVH (p=0.029), the values of which did not differ statistically. It rose with LVH degree (not significantly, p=0.217). There were no significant differences in systolic pressure among study groups (p=0.177). We concluded that pulse pressure had the greatest impact on the left ventricular geometry, particularly of the concentric type.

- Paralumbar muscle performance and fatigue were evaluated by measuring electromagnetic activity during entire body vibration (EBV) in 44 healthy subjects. Physical fitness of subjects was estimated on a 5-degree scale. Electric activity was recorded in 200 seconds with 1 kHz sampling on the Biopac Student Lab during EBV. Data were used to produce time series for two vectors of the phase space and spatial axis: X (left-right), Y (up-down) and Z (ventral-dorsal). Time series were evaluated by calculating fractal dimension by the R/S algorithm. Movement of the electric field along the Y-axis showed changes (up-down) extracted in the first and second quarter of the measurement (p=0.02 and p=0.03, respectively). These changes were not specific for gender but showed dependence on subject age and fitness. The fractal dimension values by the R/S algorithm were larger in female subjects. Results suggested the electric field changes during EBV in the up-down direction to contain information on muscular performance and fatigue, not dependent on gender, but on the age and degree of overall physical fitness.

Background: INTERBIO-21 st is Phase II of the INTERGROWTH-21 st Project, the population-based, research initiative involving nearly 70,000 mothers and babies worldwide coordinated by Oxford University and performed by a multidisciplinary network of more than 400 healthcare professionals and scientists from 35 institutions in 21 countries worldwide. Phase I, conducted 2008-2015, consisted of nine complementary studies designed to describe optimal human growth and neurodevelopment, based conceptually on the WHO prescriptive approach. The studies generated a set of international standards for monitoring growth and neurodevelopment, which complement the existing WHO Child Growth Standards. Phase II aims to improve the functional classification of the highly heterogenous preterm birth and fetal growth restriction syndromes through a better understanding of how environmental exposures, clinical conditions and nutrition influence patterns of human growth from conception to childhood, as well as specific neurodevelopmental domains and associated behaviors at 2 years of age. Methods: In the INTERBIO-21 st Newborn Case-Control Study, a major component of Phase II, our objective is to investigate the mechanisms potentially responsible for preterm birth and small for gestational age and their interactions, using deep phenotyping of clinical, growth and epidemiological data and associated nutritional, biochemical, omic and histological profiles. Here we describe the study sites, population characteristics, study design, methodology and standardization procedures for the collection of longitudinal clinical data and biological samples (maternal blood, umbilical cord blood, placental tissue, maternal feces and infant buccal swabs) for the study that was conducted between 2012 and 2018 in Brazil, Kenya, Pakistan, South Africa, Thailand and the UK. Discussion: Our study provides a unique resource for the planned analyses given the range of potentially disadvantageous exposures (including poor nutrition, pregnancy complications and infections) in geographically diverse populations worldwide. The study should enhance current medical knowledge and provide new insights into environmental influences on human growth and neurodevelopment.

Evidence suggests that high-level physical activity may potentially reduce cancer mortality through its immune enhancement effect. We therefore hypothesized that survival benefits associated with physical activity might be stronger in colorectal carcinomas with lower immune reaction at diagnosis.

Background: Suicide ideation is a health issue affecting adolescents worldwide. There are significant variations in suicide ideation between countries and genders, which have not been fully explained. Research is especially lacking in countries outside Europe and North America. Gender equality has been shown to matter in other aspects of adolescent mental health, such as life satisfaction, but has not been researched in relation to suicide ideation at national level. Objective: To investigate how national gender inequality is related to self-reported suicide ideation among adolescents, and whether this association differs between boys and girls. Methods: This is a cross-national, cross-sectional study using individual survey data from the Global School-based Student Health Survey, a survey in Africa, Asia, Latin America and the South Pacific, developed and supported by among others the WHO and the CDC; connecting this to national data: the gender inequality index from the UNDP; controlling for GDP per capita and secondary school enrolment. The data was analysed using a multilevel logistic regression method and included 149,306 students from 37 countries. Results: Higher national gender inequality, as measured by the gender inequality index, was significantly associated with a higher likelihood of suicide ideation in both girls and boys (odds ratio: 1.38 p-value: 0.015), but for girls and both sexes this was only after adjusting for selection bias due to secondary school enrolment (as well as GDP/capita). Interaction models showed that this association was stronger in boys than in girls. Conclusions: National gender inequality seems to be associated with higher levels of suicide ideation among adolescents in mainly low- and middle-income countries, especially among boys.

CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 "antigen sink". A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8+ T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.

Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 Å resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules.

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not achievable with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated, but as many of them are small recombinant fragments, their utility could be limited. For some therapeutic applications, full-size IgGs may be the optimal format. Two challenges should be met to make bispecific IgGs; one is that each heavy chain will only pair with the heavy chain of the second specificity and that homodimerization be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and not with the light chain of the second specificity. The first solution to the first criterion (knobs into holes, KIH) was presented in 1996 by Paul Carter's group from Genentech. Additional solutions were presented later on. However, until recently, out of >120 published bsAb formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested. We present a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL. We name antibodies produced according to this design "BIClonals". Bispecific IgGs where the artificial disulfide bond is placed in the CH1-CL interface are also presented. Briefly, we found that an artificial disulfide bond between VH position 44 to VL position 100 provides for effective and correct H-L chain pairing while also preventing the formation of wrong H-L chain pairs. When the artificial disulfide bond links the CH1 with the CL domain, effective H-L chain pairing also occurs, but in some cases, wrong H-L pairing is not totally prevented. We conclude that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies, hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs, making it necessary to carefully evaluate the optimal solution for each new antibody.

Monoclonal antibody therapeutics have proven to be successful treatment options for patients in various indications. Particularly in oncology, therapeutic concepts involving antibodies often rely on the so-called effector functions, such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), which are programed in the antibody Fc region. However, Fc-mediated effector mechanisms often seem to be insufficient in properly activating the immune system to act against tumor cells. Furthermore, long term treatments can lead to resistance against the applied drug, which is monospecific by nature. There is promise in using specific antibodies to overcome such issues due to their capability of recruiting and activating T-cells directly at the tumor site, for instance. During the last decade, two of these entities, which are referred to as Blinatumomab and Catumaxomab, have been approved to treat patients with acute lymphoblastic leukemia and malignant ascites. In addition, Emicizumab, which is a bispecific antibody targeting clotting factors IXa and X, was recently granted market approval by the FDA in 2017 for the treatment of hemophilia A. However, the generation of these next generation therapeutics is challenging and requires tremendous engineering efforts as two distinct paratopes need to be combined from two different heavy and light chains. This mini review summarizes technologies, which enable the generation of antibodies with dual specificities.

Bispecific antibodies represent an emerging class of antibody drugs that are commonly generated by fusion of Fv or scFv antigen binding domains to IgG or Fab scaffolds. Fv- or scFv-mediated multimerisation of bispecific antibodies via promiscuous vH-vL pairing can result in sub-optimal monomer levels during expression, and hence, undesirable therapeutic protein yields. We investigate the contribution of disulphide stabilised Fv and scFv to Fab-Fv and Fab-scFv multimerisation. We show that monomer levels of isolated Fv/scFv cannot always be used to predict monomer levels of Fab-linked Fv/scFv, and that Fab-scFv monomer levels are greater than the equivalent Fab-Fv. Through grafting bispecifics with framework/CDR-'swapped' Fv and scFv, we show that monomer levels of disulphide stabilised Fab-Fv and Fab-scFv can be improved by Fv framework 'swapping'. The Fab-Fv and Fab-scFv can be considered representative of the significant number of bispecific antibody formats containing appended Fv/scFv, as we also used Fv framework 'swapping' to increase the monomer level of an IgG-scFv bispecific antibody. This research may, therefore, be useful for maximising the monomeric yield of numerous pharmaceutically-relevant bispecific formats in pre-clinical development.

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

Background: Dimethyl sulfoxide (DMSO) has been used for medical treatment and as a pharmacological agent in humans since the 1960s. Today, DMSO is used mostly for cryopreservation of stem cells, treatment of interstitial cystitis, and as a penetrating vehicle for various drugs. Many adverse reactions have been described in relation to the use of DMSO, but to our knowledge, no overview of the existing literature has been made. Our aim was to conduct a systematic review describing the adverse reactions observed in humans in relation to the use of DMSO. Methods: This systematic review was reported according to the PRISMA-harms (Preferred Reporting Items for Systematic reviews and Meta-Analysis) guidelines. The primary outcome was any adverse reactions occurring in humans in relation to the use of DMSO. We included all original studies that reported adverse events due to the administration of DMSO, and that had a population of five or more. Results: We included a total of 109 studies. Gastrointestinal and skin reactions were the commonest reported adverse reactions to DMSO. Most reactions were transient without need for intervention. A relationship between the dose of DMSO given and the occurrence of adverse reactions was seen. Conclusions: DMSO may cause a variety of adverse reactions that are mostly transient and mild. The dose of DMSO plays an important role in the occurrence of adverse reactions. DMSO seems to be safe to use in small doses. Registration: PROSPERO CRD42018096117.

Background: Commercially available conventional growth media for the culture of microbes are expensive, hence the need for alternative cheaper sources. Livestock waste, in the form of feather and blood, are of value in biotechnology because of their high protein content. Hence the primary aim of this study was to produce a cheaper peptone alternative from chicken feather protein hydrolysate (CFPH) and blood meal (BM). Methods: The growth of selected bacteria and fungi was monitored in different media prepared from varied concentrations of peptone, CFPH and BM in order to determine the combination that produced maximum growth. Five different media, namely 100% peptone (control), 100% BM, 40% peptone + 60% CFPH, 40% BM + 60% CFPH and 20% peptone + 20% BM + 60% CFPH were prepared and used for the study. The different media were inoculated with 1 ml of each test organism ( Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida carpophila, Candida tropicalis and Pichia kundriavzevii) and their growth monitored for 10 h. Results:Pseudomonas aeruginosa, Proteus mirabilis and Staphylococcus aureus grew best in the 100% peptone, Klebsiella pneumoniae grew best in 100 BM. The fungi species were observed to grow best in 100% peptone. The 60% CFPH + 40% peptone combination (CFPH obtained with precipitate of trichloroacetic acid (TCA), hydrochloric acid (HCl) and nitric acid (HNO 3) gave the best growth of E. coli.  The 60% CFPH + 40% peptone combination (CFPH obtained with precipitate of TCA) also gave the best growth of C. tropicalis and Klebsiella pneumoniae. Conclusions: Overall, the 60% CFPH + 40% peptone combination showed the most potential as an alternative to peptone, especially for E. coli.

Sensitive laser desorption/ionization obtained via a sublimation-coated 1,5-diaminonaphthalene (1,5-DAN) matrix allowed dual polarity MALDI-IMS analysis on the same pixel points across the jejunal mucosal region in rat small intestine which yielded high-spatial-resolution (10 μm) ion images of several lipid species correlated with the same histological features.

Adipose- derived stem cells (ADSCs) are widely used for tissue engineering and regenerative medicine. The beneficial effects of ADSCs on wound healing have already been reported. Remodeling of extracellular matrix (ECM) is the most important physiological event during wound healing. ECM is sensitive to mechanical stresses and the expression of its components can be therefore influenced. The aim of this study was to investigate the effect of simulated microgravity on gene expression of some ECM and adhesion molecules in human ADSCs. After isolation and characterization of ADSCs, cells were exposed to simulated microgravity for 1, 3 and 7 days. Real-time PCR, fluorescence immunocytochemistry, and MTT assay were performed to evaluate the alterations of integrin subunit beta 1 (ITGB1), collagen type 3 (ColIII), matrix metalloproteinase-1 (MMP1), CD44, fibrillin (FBN1), vimentin (VIM) genes, and ColIII protein levels as well as cells viability. Microgravity simulation increased the expression of ITGB1, ColIII, MMP1, and CD44 and declined the expression of FBN1 and VIM genes. ColIII protein levels also increased. There were no significant changes in the viability of cells cultured in microgravity. Since the high expression of ECM components is known as one of the fibroblast markers, our data suggest that pretreatment of ADSCs by simulated microgravity may increase their differentiation capacity towards fibroblastic cells. Microgravity had not adversely affected the viability of ADSCs, and it is likely to be used alone or in combination with biochemical inducers for cell manipulation.