The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 and eSD2 was further validated using three model proteins-glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.

Malaria control program in the Arabian Peninsula, backed by adequate logistical support, has interrupted transmission with exception of limited sites in Saudi Arabia and sporadic outbreaks in Oman. However, sustained influx of imported malaria represents a direct threat to the above success. Here we examined the extent of genetic diversity among imported P. vivax in Qatar, and its ability to produce gametocytes, compared to parasites in main sites of imported cases, the Indian subcontinent (india) and East Africa (Sudan and Ethiopia). High diversity was seen among imported P. vivax in Qatar, comparable to parasites in the Indian subcontinent and East Africa. Limited genetic differentiation was seen among imported P. vivax, which overlapped with parasites in India, but differentiated from that in Sudan and Ethiopia. Parasite density among imported cases, ranged widely between 26.25-7985934.1 Pv18S rRNA copies/µl blood, with a high prevalence of infections carried gametocytes detectable by qRT-PCR. Parasitaemia was a stronger predictor for P. vivax gametocytes density (r = 0.211, P = 0.04). The extensive diversity of imported P. vivax and its ability to produce gametocytes represent a major threat for re-introduction of malaria in Qatar. The genetic relatedness between P. vivax reported in Qatar and those in India suggest that elimination strategy should target flow and dispersal of imported malaria into the region.

As a consequence of exposure to environmental toxicants, a "field cancerization" effect occurs in the lung, resulting in the development of a field of initiated, but morphologically normal appearing cells within a damaged epithelium containing mutations in oncogene or tumor suppressor genes. Unlike humans, whose airway field of injury associated with lung cancer has long been investigated with airway brushings obtained via bronchoscopy, no methods are available for similar studies in the mouse due to the small size of the murine airways. In this protocol, we describe a detailed method for performing airway brushing from a live mouse, which enables repeated sampling from the same mouse and thus, mimicking the bronchoscopy protocol used in humans. Using this approach in the N-nitroso-tris-chloroethylurea (NTCU)-induced mouse lung squamous cell carcinoma (SCC) model, we isolated airway epithelial cells with intact cell membrane structure and then performed transcriptome sequencing (RNA-Seq). We found activation of the PI3K signaling network to be the most significant in cytologically normal bronchial airway epithelial cells of mice with preneoplastic lung SCC lesions. Prolonged exposure to NTCU also induced activation of NF-kappaB (NFƙB), the downstream pathway of PI3K; this NTCU-induced lung SCC progression can be reversed by blocking the NFƙB pathway.

Cancer-associated fibroblasts (CAFs) interact closely with cancer cells, supporting their growth and invasion. To investigate the role of microRNA-21 (miR-21) in lung adenocarcinoma, and especially in its CAF component, in situ hybridisation was applied to samples from 89 invasive lung adenocarcinoma cases. MiR-21 expression was observed in both cancer cells and CAFs. When the patients were stratified by expression, miR-21 levels in CAFs (n = 9), but not in cancer cells (n = 21), were inversely correlated with patient survival; patients with miR-21high CAFs exhibited lower survival than those with miR-21low CAFs. The underlying mechanism was investigated in vitro. Conditioned medium (CM) from A549 lung cancer cells increased miR-21 expression in MRC-5 and IMR-90 lung fibroblasts through the transforming growth factor-β pathway, and induced CAF-like morphology and migratory capacity. MiR-21 up-regulation in lung fibroblasts induced a novel CAF-secreted protein, calumenin, as well as known CAF markers (periostin, α-smooth muscle actin, and podoplanin). Moreover, CM from the lung fibroblasts increased A549 cell proliferation in a calumenin-dependent manner. Thus, miR-21 expression in lung fibroblasts may trigger fibroblast trans-differentiation into CAFs, supporting cancer progression. Therefore, CAF miR-21 represents a pivotal prognostic marker for this scar-forming cancer of the lungs.

The spiny mouse, Acomys cahirinus, is an adult mammal capable of remarkable feats of scar-free tissue regeneration after damage to several organs including the skin and the heart. Here we investigate the regenerative properties of the skeletal muscle of A. cahirinus tibialis anterior in comparison to the lab mouse, Mus musculus. The A. cahirinus TA showed a similar distribution of myosin heavy chain fibre types and a reduced proportion of oxidative fibres compared to M. musculus. There were differences in the matrix components of the TA with regard to collagen VI and the biomechanical properties. A. cahirinus TA regenerated faster with a more rapid induction of embryonic myosin and higher levels of dystrophin than in M. musculus fibres. There were lower levels of inflammation (NF-kB), fibrosis (TGFβ-1, collagens) and higher levels of the anti-inflammatory cytokine Cxcl12. There was a difference in macrophage profile between the two species. After multiple rounds of muscle regeneration the M. musculus TA failed to regenerate muscle fibres and instead produced a large numbers of adipocytes whereas the A. cahirinus TA regenerated perfectly. This clearly improved regeneration performance can be explained by differing levels of growth factors such as adiponectin between the two species.

In dicots, the key developmental process by which immature plastids differentiate into photosynthetically competent chloroplasts commences in the shoot apical meristem (SAM), within the shoot apex. Using laser-capture microdissection and single-cell RNA sequencing methodology, we studied the changes in the transcriptome along the chloroplast developmental pathway in the shoot apex of tomato seedlings. The analysis revealed the presence of transcripts for different chloroplast functions already in the stem cell-containing region of the SAM. Thereafter, an en masse up-regulation of genes encoding for various proteins occurs, including chloroplast ribosomal proteins and proteins involved in photosynthesis, photoprotection and detoxification of reactive oxygen species. The results highlight transcriptional events that operate during chloroplast biogenesis, leading to the rapid establishment of photosynthetic competence.

Antiretroviral therapy during the earliest stage of acute HIV infection (Fiebig I) might minimize establishment of a latent HIV reservoir and thereby facilitate viremic control after analytical treatment interruption. We show that 8 participants, who initiated treatment during Fiebig I and were treated for a median of 2.8 years, all experienced rapid viral load rebound following analytical treatment interruption, indicating that additional strategies are required to control or eradicate HIV.

Evidence from mouse chronic viral infection models suggests that CD8+ T cell subsets characterized by distinct expression levels of the receptor PD-1 diverge in their state of exhaustion and potential for reinvigoration by PD-1 blockade. However, it remains unknown whether T cells in human cancer adopt a similar spectrum of exhausted states based on PD-1 expression levels. We compared transcriptional, metabolic and functional signatures of intratumoral CD8+ T lymphocyte populations with high (PD-1T), intermediate (PD-1N) and no PD-1 expression (PD-1-) from non-small-cell lung cancer patients. PD-1T T cells showed a markedly different transcriptional and metabolic profile from PD-1N and PD-1- lymphocytes, as well as an intrinsically high capacity for tumor recognition. Furthermore, while PD-1T lymphocytes were impaired in classical effector cytokine production, they produced CXCL13, which mediates immune cell recruitment to tertiary lymphoid structures. Strikingly, the presence of PD-1T cells was strongly predictive for both response and survival in a small cohort of non-small-cell lung cancer patients treated with PD-1 blockade. The characterization of a distinct state of tumor-reactive, PD-1-bright lymphocytes in human cancer, which only partially resembles that seen in chronic infection, provides potential avenues for therapeutic intervention.

To evaluate the effects of HCV NS5B amino acid substitutions on treatment outcome in Ledipasvir (LDV)/Sofosbuvir (SOF) for Japanese patients with genotype 1b HCV infection, NS5B sequences were examined in i) seven patients experiencing virologic failure after LDV/SOF in real-world practice, ii) 109 SOF-naïve patients, iii) 165 patients enrolled in Phase-3 LDV/SOF trial. A218S and C316N were detected in all patients with viral relapse; the percentages of these substitutions in SOF-naïve patients were 64.2% and 55.0%, respectively. Genotype 1b HCV strains with NS5B-C316N mutation were located in the leaves different from those in which HCV strains without such substitutions were present on the phylogenetic tree. Structural modeling revealed that amino acid 218 was located on the surface of the NTP tunnel. Free energy analysis based on molecular dynamics simulations demonstrated that the free energy required to pass through the tunnel was larger for triphosphate SOF than for UTP in NS5B polymerase carrying A218S, but not in wild-type. However, no susceptibility change was observed for these substitutions to SOF in replicon assay. Furthermore, the SVR rate was 100% in patients enrolled the Phase-3 trial. In conclusion, NS5B A218S and C316N were detected in all patients who relapsed following LDV/SOF in real-world practice. These substitutions did not impact the overall SVR rate after LDV/SOF, however, further studies are needed to elucidate the impact of these substitutions.

The pomegranate peel extract is a rich source of natural antioxidant and antimicrobial activity. The aim of the present investigation was to evaluate the in vivo antifungal activity of the pomegranate peel extract and to compare it with nystatin against oral candidiasis in Wistar rats.

The purpose was to clarify the effects of Helicobacter pylori (H. pylori) eradication on the changes in serum lipid levels by comparing subjects with and without continuous H. pylori infection. The study subjects were 774 individuals (males 536, females 238, mean age 52.6 years) who visited between April 2013 and March 2016 for annual medical checkups. Serum total cholesterol, high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC), and triglyceride levels, and LDLC/HDLC ratio were compared between the subjects with and without H. pylori infection, as well as those with H. pylori eradication subjects. The HDLC level in the H. pylori-positive group was significantly lower as compared to the H. pylori-negative group. The serum level of HDLC in subjects with successful eradication of H. pylori tended to be higher, while the serum levels of total cholesterol, LDLC, and triglycerides tended to be lower in comparison to subjects with continuous H. pylori infection. In addition, the LDLC/HDLC ratio in the H. pylori-positive group was significantly higher than that in the H. pylori-negative group, and successful H. pylori eradication tended to reduce that ratio. In conclusion, successful eradication of H. pylori may have favorable effects on lipid metabolism.

Although chronic constipation is common, colonic functional evaluating tests are uncommon. This study examines whether chronic constipation and gastrointestinal symptoms are correlated with the lateral diameter of the colon measured from MRI images. We included chronic constipation patients in a prospective, cross-sectional study using MRI at three centers. We divided 3D MRI colorectal images into 6 segments using with specified sequences and selected the maximum luminal diameter from each segment. We used the GSRS questionnaire to evaluate gastrointestinal symptoms. We evaluated the correlation between luminal diameters and GSRS scores. We found the following positive correlations: descending colon and unsatisfactory defecation symptoms; sigmoid colon and diarrhea; and rectum and constipation. The sum and ratio of the ascending and sigmoid colon diameters correlated with nausea and diarrhea. The sum of the transvers to the sigmoid colon diameter also correlated with nausea and diarrhea. The sum of all segment diameters correlated with nausea and constipation. In conclusion, we showed cross-sectional study of colonic MRI correlate with gastrointestinal symptoms. MRI might be useful for colonic motility evaluations to determine appropriate constipation treatments (Clinical trial registry number UMIN 000021274).

It is becoming evident that microglia, the resident immune cells of the central nervous system (CNS), are active contributors in neurological disorders. Nevertheless, the impact of microgliosis on neuropathology, behavior and clinical decline in neuropathological conditions remains elusive. A mouse model lacking multifunctional protein-2 (MFP2), a pivotal enzyme in peroxisomal β-oxidation, develops a fatal disorder characterized by motor problems similar to the milder form of human disease. The molecular mechanisms underlying neurological decline in men and mice remain unknown. The hallmark of disease in the mouse model is chronic proliferation of microglia in the brain without provoking neuronal loss or demyelination. In order to define the contribution of Mfp2-/- neural cells to development of microgliosis and clinical neuropathology, the constitutive Mfp2-/- mouse model was compared to a neural selective Nestin-Mfp2-/- mouse model. We demonstrate in this study that, in contrast to early-onset and severe microgliosis in constitutive Mfp2-/- mice, Mfp2+/+ microglia in Nestin-Mfp2-/- mice only become mildly inflammatory at end stage of disease. Mfp2-/- microglia are primed and acquire a chronic and strong inflammatory state in Mfp2-/- mice whereas Mfp2+/+ microglia in Nestin-Mfp2-/- mice are not primed and adopt a minimal activation state. The inflammatory microglial phenotype in Mfp2-/- mice is correlated with more severe neuronal dysfunction, faster clinical deterioration and reduced life span compared to Nestin-Mfp2-/- mice. Taken together, our study shows that deletion of MFP2 impairs behavior and locomotion. Clinical decline and neural pathology is aggravated by an early-onset and excessive microglial response in Mfp2-/- mice and strongly indicates a cell-autonomous role of MFP2 in microglia.

Diverse animal species perceive Earth's magnetism and use their magnetic sense to orientate and navigate. Even non-migrating insects such as fruit flies and cockroaches have been shown to exploit the flavoprotein Cryptochrome (Cry) as a likely magnetic direction sensor; however, the transduction mechanism remains unknown. In order to work as a system to steer insect flight or control locomotion, the magnetic sense must transmit the signal from the receptor cells to the brain at a similar speed to other sensory systems, presumably within hundreds of milliseconds or less. So far, no electrophysiological or behavioral study has tackled the problem of the transduction delay in case of Cry-mediated magnetoreception specifically. Here, using a novel aversive conditioning assay on an American cockroach, we show that magnetic transduction is executed within a sub-second time span. A series of inter-stimulus intervals between conditioned stimuli (magnetic North rotation) and unconditioned aversive stimuli (hot air flow) provides original evidence that Cry-mediated magnetic transduction is sufficiently rapid to mediate insect orientation.

In schizophrenia (SCZ), dysfunction of the dorsolateral prefrontal cortex (DLPFC) has been linked to the deficits in executive functions and attention. It has been suggested that, instead of considering the right DLPFC as a cohesive functional entity, it can be divided into two parts (anterior and posterior) based on its whole-brain connectivity patterns. Given these two subregions' differential association with cognitive processes, we investigated the functional connectivity (FC) profile of both subregions through resting-state data to determine whether they are differentially affected in SCZ. Resting-state magnetic resonance imaging (MRI) scans were obtained from 120 patients and 172 healthy controls (HC) at 6 different MRI sites. The results showed differential FC patterns for the anterior and posterior parts of the right executive control-related DLPFC in SCZ with the parietal, the temporal and the cerebellar regions, along with a convergent reduction of connectivity with the striatum and the occipital cortex. An increased psychopathology level was linked to a higher difference in posterior vs. anterior FC for the left IFG/anterior insula, regions involved in higher-order cognitive processes. In sum, the current analysis demonstrated that even between two neighboring clusters connectivity could be differentially disrupted in SCZ. Lacking the necessary anatomical specificity, such notions may in fact be detrimental to a proper understanding of SCZ pathophysiology.

Background: The Tower of London (TOL) task is one of the most commonly used tests for evaluating executive functions, and can indicate planning and problem-solving abilities. The aim of this study was to evaluate hemodynamic changes between the task period and rest period in patients with bipolar depression during the TOL task and the verbal fluency task (VFT) using near-infrared spectroscopy (NIRS). Methods: Forty-three patients with bipolar depression and 32 healthy controls (HCs) matched for sex, age, handedness, and years of education were enrolled in this study. All participants were aged between 16 and 50. All patients in our study were taking medications such as antidepressants, antipsychotics and mood stabilizers at the time of measurement. Changes in oxygenated hemoglobin (oxy-Hb) levels in frontal areas during the TOL task and VFT were evaluated using a 41-channel NIRS system. Results: During the TOL task, the patients with bipolar depression exhibited significantly smaller changes in the bilateral dorsal-lateral prefrontal cortex (DLPFC) than the HCs. During the VFT task, the patients with bipolar depression exhibited significantly smaller changes in the right ventrolateral prefrontal cortex (VLPFC), the right DLPFC and both the right and left prefrontal cortex (PFC) than the HCs. Limitations: Our sample size was small, and the effects of medication cannot be excluded. Conclusions: These results indicate that planning and problem solving dysfunction is related to the impairment of the prefrontal cortex in patients with bipolar depression, and NIRS can be used to assess planning and problem solving abilities, which are essential to daily life in patients with bipolar disorder.

Due to the high prevalence in pregnant women and potential association with pregnancy complications or perinatal outcomes, sleep-disordered breathing (SDB) has become an increasing concern.

Alterations in maternal thyroid physiology may have deleterious consequences on the development of the fetal brain, but the underlying mechanisms remain elusive, hampering the development of appropriate therapeutic strategies. The present review sums up the contribution of genetically modified mouse models to this field. In particular, knocking out genes involved in thyroid hormone (TH) deiodination, transport, and storage has significantly improved the picture that we have of the economy of TH in the fetal brain and the underlying genetic program. These data pave the way for future studies to bridge the gap in knowledge between thyroid physiology and brain development.

Using animal models, the gut microbiota has been shown to play a critical role in the health and disease of many organ systems. Unfortunately, animal model studies often lack reproducibility when performed at different institutions. Previous studies in our laboratory have shown that the gut microbiota of mice can vary with a number of husbandry factors leading us to speculate that differing environments may alter gut microbiota, which in turn may influence animal model phenotypes. As an extension of these studies, we hypothesized that the shipping of mice from a mouse producer to an institution will result in changes in the type, relative abundance, and functional composition of the gut microbiota. Furthermore, we hypothesized that mice will develop a microbiota unique to the institution and facility in which they are housed. To test these hypotheses, mice of two strains (C57BL/6J and BALB/cJ), two age groups (4 week and 8 week old), and originating from two types of housing (research animal facility under conventional housing and production facilities under maximum barrier housing) were obtained from The Jackson Laboratory. Fecal samples were collected the day prior to shipping, immediately upon arrival, and then on days 2, 5, 7, and weeks 2, 4, and 9 post-arrival. Following the first post-arrival fecal collection, mice were separated into 2 groups and housed at different facilities at our institution while keeping their caging, diet, and husbandry practices the same. DNA was extracted from the collected fecal pellets and 16S rRNA amplicons were sequenced in order to characterize the type and relative abundance of gut bacteria. Principal component analysis (PCA) and permutational multivariate analysis of variance (PERMANOVA) demonstrated that both the shipping and the institution and facility in which mice were housed altered the gut microbiota. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) predicted differences in functional composition in the gut microbiota of mice based on time of acclimation.

Effector CD8+ T cells generally produce type-1 cytokines and mediators of the perforin/granzyme cytolytic pathway, yet type-2-polarized CD8+ cells (Tc2) are detected in type-2 (T2) cytokine-driven diseases such as asthma. It is unclear whether T2 cytokine exposure during activation is sufficient to polarize human CD8+ T cells. To address this question, a protocol was developed for high-efficiency activation of human CD8+ T cells in which purified single cells or populations were stimulated with plate-bound anti-CD3 and anti-CD11a mAb for up to 8 days in T2 polarizing or neutral conditions, before functional analysis. Activation of CD8+ naïve T cells (TN) in T2 compared with neutral conditions decreased the size of single-cell clones, although early division kinetics were equivalent, indicating an effect on overall division number. Activation of TN in T2 conditions followed by brief anti-CD3 mAb restimulation favored expression of T2 cytokines, GATA3 and Eomes, and lowered expression of type-1 cytokines, Prf1, Gzmb, T-BET, and Prdm1. However, IL-4 was only weakly expressed, and PMA and ionomycin restimulation favored IFN-γ over IL-4 expression. Activation of TN in T2 compared with neutral conditions prevented downregulation of costimulatory (CD27, CD28) and lymph-node homing receptors (CCR7) and CD95 acquisition, which typically occur during differentiation into effector phenotypes. CD3 was rapidly and substantially induced after activation in neutral, but not T2 conditions, potentially contributing to greater division and differentiation in neutral conditions. CD8+ central memory T cells (TCM) were less able to enter division upon reactivation in T2 compared with neutral conditions, and were more refractory to modulating IFN-γ and IL-4 production than CD8+ TN. In summary, while activation of TN in T2 conditions can generate T2 cytokine-biased cells, IL-4 expression is weak, T2 bias is lost upon strong restimulation, differentiation, and division are arrested, and reactivation of TCM is reduced in T2 conditions. Taken together, this suggests that exposure to T2 cytokines during activation may not be sufficient to generate and retain human Tc2 cells.