Peptidoglycan recognition in Drosophila is mediated by LysMD3/4.

Microbial recognition is a key step in regulating the immune signaling pathways of multicellular organisms. Peptidoglycan, a component of the bacterial cell wall, exhibits immune stimulating activity in both plants and animals. Lysin motif domain (LysMD) family proteins are ancient peptidoglycan receptors that function in bacteriophage and plants. This report focuses on defining the role of LysMD-containing proteins in animals. Here, we characterize a novel transmembrane LysMD family protein. Loss-of-function mutations at the lysMD3/4 locus in Drosophila are associated with systemic innate immune activation following challenge, so we refer to this gene as immune active (ima). We show that Ima selectively binds peptidoglycan, is enriched in cell membranes, and is necessary to regulate terminal innate immune effectors through an NF-kB-dependent pathway. Hence, Ima fulfills the key criteria of a peptidoglycan pattern recognition receptor. The human Ima ortholog, hLysMD3, exhibits similar biochemical properties. Together, these findings establish LysMD3/4 as the founding member of a novel family of animal peptidoglycan recognition proteins.

Current transfusion practice and need for new blood products to ensure blood supply for patients with major hemorrhage in Europe.

BACKGROUND: New blood products are considered for treatment of patients with major hemorrhage. The aim of this report is to describe the current transfusion practices in Europe for patients with major hemorrhage and explore the need for new or modified blood products to ensure prehospital and in-hospital blood supply. STUDY DESIGN AND METHOD: The European Blood Alliance (EBA) Working Group on Innovation and New Blood Products' subgroup on major hemorrhage performed a survey among the EBA member states. RESULTS: The response rate was 58% (17 responses from 15 of the 26 EBA member states). Of these, sixteen (94%) provide massive transfusion packages (MTPs) with balanced ratio of red blood cells and plasma. Seven of the respondents included platelets from the start of treatment. Eleven (65%) provide prehospital blood products, mainly red cell concentrates or dried and/or thawed plasma with 5 days of extended storage. Two countries provide prehospital whole blood. Twelve respondents (71%) saw a need for implementation of new or modified blood components in their institution. The top three priorities were whole blood (12 of 12, 100%), dried plasma (8 of 12, 67%), and cold-stored platelets (7 of 12, 58%). DISCUSSION: Current national guidelines for use of blood products in patients with major hemorrhage in Europe agree on the use of balanced transfusion, however the timing and source of platelets differ. Blood products for prehospital transfusion are available in several European countries. An interest in new or modified blood products for patients with major hemorrhage was observed, especially for whole blood.

Recommendations for in vitro evaluation of blood components collected, prepared and stored in non-DEHP medical devices.

BACKGROUND AND OBJECTIVES: DEHP, di(2-ethylhexyl) phthalate, is the most common member of the class of ortho-phthalates, which are used as plasticizers. The Medical Device Regulation has restricted the use of phthalates in medical devices. Also DEHP has been added to the Annex XIV of REACH, "Registration, Evaluation, Authorisation and Restriction of Chemicals" due to its endocrine disrupting properties to the environment. As such, the sunset date for commercialisation of DEHP-containing blood bags is May 27th 2025. There are major concerns in meeting this deadline as these systems have not yet been fully validated and/or CE-marked. Also, since DEHP is known to affect red cell quality during storage, it is imperative to transit to non-DEHP without affecting blood product quality. Here, EBA members aim to establish common grounds on the evaluation and assessment of blood components collected, prepared and stored in non-DEHP devices. MATERIALS AND METHODS: Based on data as well as the input of relevant stakeholders a rationale for the validation of each component was composed. RESULTS: The red cell components will require the most extensive validation as their quality is directly affected by the absence of DEHP, as opposed to platelet and plasma components. CONCLUSION: Studies in the scope of evaluating the quality of blood products obtained with non-DEHP devices, under the condition that they are carried out according to these recommendations, could be used by all members of the EBA to serve as scientific support in the authorization process specific to their jurisdiction or for their internal validation use.

Selective sp3 C-H alkylation via polarity-match-based cross-coupling.

The functionalization of carbon-hydrogen (C-H) bonds is one of the most attractive strategies for molecular construction in organic chemistry. The hydrogen atom is considered to be an ideal coupling handle, owing to its relative abundance in organic molecules and its availability for functionalization at almost any stage in a synthetic sequence. Although many C-H functionalization reactions involve C(sp3)-C(sp2) coupling, there is a growing demand for C-H alkylation reactions, wherein sp3 C-H bonds are replaced with sp3 C-alkyl groups. Here we describe a polarity-match-based selective sp3 C-H alkylation via the combination of photoredox, nickel and hydrogen-atom transfer catalysis. This methodology simultaneously uses three catalytic cycles to achieve hydridic C-H bond abstraction (enabled by polarity matching), alkyl halide oxidative addition, and reductive elimination to enable alkyl-alkyl fragment coupling. The sp3 C-H alkylation is highly selective for the α-C-H of amines, ethers and sulphides, which are commonly found in pharmaceutically relevant architectures. This cross-coupling protocol should enable broad synthetic applications in de novo synthesis and late-stage functionalization chemistry.

Ultra-low frequency labeling of single cell lineages in Drosophila.

We describe a new methodology for genetically labeling single cell lineages in Drosophila called DMARCM. The system offers ultra-low frequency labeling, linear induction, consistent labeling among individuals and virtually no background signal. We compare this technique to an existing approach, which has been widely adopted. We demonstrate how application of DMARCM in the gastrointestinal epithelium permits the effects of labeling frequency on tumorigenic stem cell growth to be distinguished in an established tumor model.

Decarboxylative Trifluoromethylation of Aliphatic Carboxylic Acids.

Herein we disclose an efficient method for the conversion of carboxylic acids to trifluoromethyl groups via the combination of photoredox and copper catalysis. This transformation tolerates a wide range of functionality including heterocycles, olefins, alcohols, and strained ring systems. To demonstrate the broad potential of this new methodology for late-stage functionalization, we successfully converted a diverse array of carboxylic acid-bearing natural products and medicinal agents to the corresponding trifluoromethyl analogues.

Transplantation of hepatitis C virus (HCV) antibody positive, nucleic acid test negative donor kidneys to HCV negative patients frequently results in seroconversion but not HCV viremia.

Anecdotal reports have suggested that transplantation of hepatitis C virus (HCV) antibody positive (Ab+)/nucleic acid test negative (NAT-) donor kidneys into HCV negative recipients is not associated with HCV transmission. We reviewed our center's outcomes of 32 HCV negative patients who received kidney allografts from 25 donors who were HCV Ab+/NAT-. The mean recipient age was 56.9 ± 12.1 years and the mean donor age was 41.5 ± 14 years, with a median Kidney Donor Profile Index (KDPI) of 68%. Twelve donors (48%) met Public Health Service (PHS) increased risk status. All patients received antithymocyte globulin induction followed by tacrolimus, mycophenolate mofetil, and steroid maintenance immunosuppression. With a mean follow-up posttransplant of 10 ± 2.7 months, 1- and 3- month serum creatinine levels were 1.7 ± 0.8 and 1.3 ± 0.4, respectively, and patient and graft survival rates were 100% and 97%, respectively. Fourteen patients (44%) seroconverted and became HCV Ab+ posttransplant. However, all 32 patients were HCV RNA negative at 1- and 3- months posttransplant, and 27 and 8 patients tested at 6- and 12-months posttransplant, respectively, remain HCV RNA negative. In conclusion, transplantation of HCV Ab+/NAT- kidneys to HCV negative recipients frequently causes HCV Ab seroconversion but not HCV viremia.

Generation of enteroendocrine cell diversity in midgut stem cell lineages.

The endocrine system mediates long-range peptide hormone signaling to broadcast changes in metabolic status to distant target tissues via the circulatory system. In many animals, the diffuse endocrine system of the gut is the largest endocrine tissue, with the full spectrum of endocrine cell subtypes not yet fully characterized. Here, we combine molecular mapping, lineage tracing and genetic analysis in the adult fruit fly to gain new insight into the cellular and molecular mechanisms governing enteroendocrine cell diversity. Neuropeptide hormone distribution was used as a basis to generate a high-resolution cellular map of the diffuse endocrine system. Our studies show that cell diversity is seen at two distinct levels: regional and local. We find that class I and class II enteroendocrine cells can be distinguished locally by combinatorial expression of secreted neuropeptide hormones. Cell lineage tracing studies demonstrate that class I and class II cells arise from a common stem cell lineage and that peptide profiles are a stable feature of enteroendocrine cell identity during homeostasis and following challenge with the enteric pathogen Pseudomonas entomophila. Genetic analysis shows that Notch signaling controls the establishment of class II cells in the lineage, but is insufficient to reprogram extant class I cells into class II enteroendocrine cells. Thus, one mechanism by which secretory cell diversity is achieved in the diffuse endocrine system is through cell-cell signaling interactions within individual adult stem cell lineages.

Receptor heterogeneity in optical biosensors.

Scientists measure rate constants associated with biochemical reactions in an optical biosensor-an instrument in which ligand molecules are convected through a flow cell over a surface to which receptors are immobilized. We quantify transport effects on such reactions by modeling the associated convection-diffusion equation with a reaction boundary condition. In experimental situations, the full PDE model reduces to a set of unwieldy integrodifferential equations (IDEs). Employing common physical assumptions, we may reduce the system to an ODE model, which is more useful in practice, and which can be easily adapted to the inverse problem of finding rate constants. The results from the ODE model compare favorably with numerical simulations of the IDEs, even outside its range of validity.

Measuring kinetic rate constants of multiple-component reactions with optical biosensors.

One may measure the kinetic rate constants associated with biochemical reactions using an optical biosensor: an instrument in which ligand molecules are convected through a flow cell over a surface to which receptors are immobilized. If there are multiple reactants, one is faced with the problem of fitting multiple kinetic rate constants to one signal, since data from all of the reacting species is lumped together. Even in the presence of ambiguous data, one may use a series of experiments to accurately determine the rate constants. Moreover, the true set of rate constants may be identified by either postprocessing the signals or adjusting the ligand inflow concentrations.

Transport Effects on Multiple-Component Reactions in Optical Biosensors.

Optical biosensors are often used to measure kinetic rate constants associated with chemical reactions. Such instruments operate in the surface-volume configuration, in which ligand molecules are convected through a fluid-filled volume over a surface to which receptors are confined. Currently, scientists are using optical biosensors to measure the kinetic rate constants associated with DNA translesion synthesis-a process critical to DNA damage repair. Biosensor experiments to study this process involve multiple interacting components on the sensor surface. This multiple-component biosensor experiment is modeled with a set of nonlinear integrodifferential equations (IDEs). It is shown that in physically relevant asymptotic limits these equations reduce to a much simpler set of ordinary differential equations (ODEs). To verify the validity of our ODE approximation, a numerical method for the IDE system is developed and studied. Results from the ODE model agree with simulations of the IDE model, rendering our ODE model useful for parameter estimation.

The Discovery of 7-Isopropoxy-2-(1-methyl-2-oxabicyclo[2.1.1]hexan-4-yl)-N-(6-methylpyrazolo[1,5-a]pyrimidin-3-yl)imidazo[1,2-a]pyrimidine-6-carboxamide (BIO-7488), a Potent, Selective, and CNS-Penetrant IRAK4 Inhibitor for the Treatment of Ischemic Stroke.

Interleukin receptor-associated kinase 4 (IRAK4) is a key node of signaling within the innate immune system that regulates the production of inflammatory cytokines and chemokines. The presence of damage-associated molecular patterns (DAMPs) after tissue damage such as stroke or traumatic brain injury (TBI) initiates signaling through the IRAK4 pathway that can lead to a feed-forward inflammatory loop that can ultimately hinder patient recovery. Herein, we describe the first potent, selective, and CNS-penetrant IRAK4 inhibitors for the treatment of neuroinflammation. Lead compounds from the series were evaluated in CNS PK/PD models of inflammation, as well as a mouse model of ischemic stroke. The SAR optimization detailed within culminates in the discovery of BIO-7488, a highly selective and potent IRAK4 inhibitor that is CNS penetrant and has excellent ADME properties.

A Tiny Pocket Packs a Punch: Leveraging Pyridones for the Discovery of CNS-Penetrant Aza-indazole IRAK4 Inhibitors.

We herein report the discovery, synthesis, and evolution of a series of indazoles and azaindazoles as CNS-penetrant IRAK4 inhibitors. Described is the use of structure-based and property-based drug design strategically leveraged to guide the property profile of a key series into a favorable property space while maintaining potency and selectivity. Our rationale that led toward functionalities with potency improvements, CNS-penetration, solubility, and favorable drug-like properties is portrayed. In vivo evaluation of an advanced analogue showed significant, dose-dependent modulation of inflammatory cytokines in a mouse model. In pursuit of incorporating a highly engineered bridged ether that was crucial to metabolic stability in this series, significant synthetic challenges were overcome to enable the preparation of the analogues.

Discovery of BIO-8169─A Highly Potent, Selective, and Brain-Penetrant IRAK4 Inhibitor for the Treatment of Neuroinflammation.

Interleukin receptor associated kinase 4 (IRAK4) plays an important role in innate immune signaling through Toll-like and interleukin-1 receptors and represents an attractive target for the treatment of inflammatory diseases and cancer. We previously reported the development of a potent, selective, and brain-penetrant imidazopyrimidine series of IRAK4 inhibitors. However, lead molecule BIO-7488 (1) suffered from low solubility which led to variable PK, compound accumulation, and poor in vivo tolerability. Herein, we describe the discovery of a series of pyridone analogs with improved solubility which are highly potent, selective and demonstrate desirable PK profiles including good oral bioavailability and excellent brain penetration. BIO-8169 (2) reduced the in vivo production of pro-inflammatory cytokines, was well tolerated in safety studies in rodents and dog at margins well above the predicted efficacious exposure and showed promising results in a mouse model for multiple sclerosis.

Simple catalytic mechanism for the direct coupling of α-carbonyls with functionalized amines: a one-step synthesis of Plavix.

The direct α-amination of ketones, esters, and aldehydes has been accomplished via copper catalysis. In the presence of catalytic copper(II) bromide, a diverse range of carbonyl and amine substrates undergo fragment coupling to produce synthetically useful α-amino-substituted motifs. The transformation is proposed to proceed via a catalytically generated α-bromo carbonyl species; nucleophilic displacement of the bromide by the amine then delivers the α-amino carbonyl adduct while the catalyst is reconstituted. The practical value of this transformation is highlighted through one-step syntheses of two high-profile pharmaceutical agents, Plavix and amfepramone.

Injury and performance related biomechanical differences between recreational and collegiate runners.

Introduction: Running related injuries (RRI) are common, but factors contributing to running performance and RRIs are not commonly compared between different types of runners. Methods: We compared running biomechanics previously linked to RRIs and performance between 27 recreational and 35 collegiate runners. Participants completed 5 overground running trials with their dominant limb striking a force plate, while outfitted with standardised footwear and 3-dimensional motion capture markers. Results: Post hoc comparisons revealed recreational runners had a larger vertical loading rate (194.5 vs. 111.5 BW/s, p < 0.001) and shank angle (6.80 vs. 2.09, p < 0.001) compared with the collegiate runners who demonstrated greater vertical impulse (0.349 vs. 0.233 BWs, p < 0.001), negative impulse (-0.022 vs. -0.013 BWs, p < 0.001), positive impulse (0.024 vs. 0.014 BWs, p < 0.001), and propulsive force (0.390 vs. 0.333 BW, p = 0.002). Adjusted for speed, collegiate runners demonstrated greater total support moment (TSM), plantar flexor moment, knee extensor moment, hip extensor moment, and had greater proportional plantar flexor moment contribution and less knee extensor moment contribution to the TSM compared with recreational runners. Unadjusted for speed, collegiate runners compared with recreational had greater TSM and plantar flexor moment but similar joint contributions to the TSM. Discussion: Greater ankle joint contribution may be more efficient and allow for greater capacity to increase speed. Improving plantarflexor function during running provides a strategy to improve running speed among recreational runners. Moreover, differences in joint kinetics and ground reaction force characteristics suggests that recreational and collegiate runners may experience different types of RRI.

Dysregulated miRNAs modulate tumor microenvironment associated signaling networks in pancreatic ductal adenocarcinoma.

The desmoplastic and complex tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) has presented tremendous challenges for developing effective therapeutic strategies. Strategies targeting tumor stroma, albeit with great potential, have met with limited success due to the lack of knowledge on the molecular dynamics within the tumor microenvironment (TME). In pursuit of a better understanding of the influence of miRNAs on TME reprogramming and to explore circulating miRNAs as diagnostic and prognostic biomarkers for PDAC, using RNA-seq, miRNA-seq, and single-cell RNA-seq (scRNA-seq), we investigated the dysregulated signaling pathways in PDAC TME modulated by miRNAs from plasma and tumor tissue. Our bulk RNA-seq in PDAC tumor tissue identified 1445 significantly differentially expressed genes with extracellular matrix and structure organization as the top enriched pathways. Our miRNA-seq identified 322 and 49 abnormally expressed miRNAs in PDAC patient plasma and tumor tissue, respectively. We found many of the TME signaling pathways were targeted by those dysregulated miRNAs in PDAC plasma. Combined with scRNA-seq from patient PDAC tumor, our results revealed that these dysregulated miRNAs were closely associated with extracellular matrix (ECM) remodeling, cell-ECM communication, epithelial-mesenchymal transition, as well as immunosuppression orchestrated by different cellular components of TME. The findings of this study could assist the development of miRNA-based stromal targeting biomarkers or therapy for PDAC patients.

Novel expression of type 1 corticotropin-releasing hormone receptor in multiple endocrine cell types in the murine anterior pituitary.

The CRH family of ligands signals via two distinct receptors, CRH-R1 and CRH-R2. Previous studies localized CRH-R1 and CRH-R2 to a subset of anterior pituitary corticotropes and gonadotropes, respectively. However, numerous studies have indicated that stress and CRH activity can alter the secretion of multiple anterior pituitary hormones, suggesting a broader expression of the CRH receptors in pituitary. To examine this hypothesis, the in vivo expression of CRH-R1 and CRH-R2 mRNA was further characterized in adult mouse pituitary. Quantitative RT-PCR analysis demonstrated that CRH-R1 mRNA is greater than 100-fold more abundant than CRH-R2 mRNA in male and female mouse pituitaries. Dual in situ hybridization analysis identified cell-specific CRH-R1 expression in the anterior pituitary. At least half of the CRH-R1-positive cells expressed proopiomelanocortin-mRNA (50% in females; 70% in males). In females, a significant percentage of the cells expressing CRH-R1 also expressed transcript for prolactin (40%), LHbeta (10%), or TSH (3%), all novel sites of CRH-R1 expression. Similarly in males, a percentage of CRH-R1-positive cells expressed prolactin (12%), LHbeta (13%), and TSH (5%). RT-PCR studies with immortalized murine anterior pituitary cell lines showed CRH-R1 and/or CRH-R2 expression in corticotropes (AtT-20 cells), gonadotropes (alphaT3-1 and LbetaT2 cells), and thyrotropes (alphaTSH cells). Whereas CRH-R1 expression in corticotropes is well established, the presence of CRH-R1 mRNA in a subset of lactotropes, gonadotropes, and thyrotropes establishes these cell types as novel sites of murine CRH-R1 expression and highlights the pituitary as an important site of interaction between the hypothalamus-pituitary-adrenal and multiple endocrine axes.

Proteasome activator PA200 is required for normal spermatogenesis.

The PA200 proteasome activator is a broadly expressed nuclear protein. Although how PA200 normally functions is not fully understood, it has been suggested to be involved in the repair of DNA double-strand breaks (DSBs). The PA200 gene (Psme4) is composed of 45 coding exons spanning 108 kb on mouse chromosome 11. We generated a PA200 null allele (PA200(Delta)) through Cre-loxP-mediated interchromosomal recombination after targeting loxP sites at either end of the locus. PA200(Delta/Delta) mice are viable and have no obvious developmental abnormalities. Both lymphocyte development and immunoglobulin class switching, which rely on the generation and repair of DNA DSBs, are unperturbed in PA200(Delta/Delta) mice. Additionally, PA200(Delta/Delta) embryonic stem cells do not exhibit increased sensitivity to either ionizing radiation or bleomycin. Thus, PA200 is not essential for the repair of DNA DSBs generated in these settings. Notably, loss of PA200 led to a marked reduction in male, but not female, fertility. This was due to defects in spermatogenesis observed in meiotic spermatocytes and during the maturation of postmeiotic haploid spermatids. Thus, PA200 serves an important nonredundant function during spermatogenesis, suggesting that the efficient generation of male gametes has distinct protein metabolic requirements.

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.