En-bloc transurethral resection vs. conventional transurethral resection for primary non-muscle invasive bladder cancer: A meta-analysis.

INTRODUCTION: We performed a meta-analysis to evaluate the effect of en-bloc transurethral resection vs. conventional transurethral resection for primary non-muscle invasive bladder cancer. METHODS: A systematic literature search up to January 2022 was done and 28 studies included 3714 primary non-muscle invasive bladder cancer subjects at the start of the study; 1870 of them were en-bloc transurethral resection, and 1844 were conventional transurethral resection for primary non-muscle invasive bladder cancer. We calculated the odds-ratio (OR) and mean-difference (MD) with 95% confidence-intervals (CIs) to evaluate the effect of en-bloc transurethral resection compared with conventional transurethral resection for primary non-muscle invasive bladder cancer by the dichotomous or continuous methods with random or fixed-effects models. RESULTS: En-bloc transurethral resection had significantly lower twenty-four-month recurrence (OR: 0.63; 95%CI: 0.50-0.78; p < 0.001), catheterization-time (MD: -0.66; 95%CI: -1.02-[-0.29]; p < 0.001), length of hospital stay (MD: -0.95; 95%CI: -1.55-[-0.34]; p = 0.002), postoperative bladder irrigation duration (MD: -6.06; 95%CI: -9.45-[-2.67]; p < 0.001), obturator nerve reflex (OR: 0.08; 95%CI: 0.02-0.34; p = 0.03), and bladder perforation (OR: 0.14; 95%CI: 0.06-0.36: p < 0.001) and no significant difference in the 12-month-recurrence (OR: 0.79; 95%CI: 0.61-1.04; p = 0.09), the operation time (MD: 0.67; 95%CI: -1.92 to 3.25; p = 0.61), and urethral stricture (OR: 0.46; 95%CI: 0.14-1.47; p = 0.0.19) compared with conventional transurethral resection for primary non-muscle invasive bladder cancer subjects. CONCLUSIONS: En-bloc transurethral resection had a significantly lower twenty-four-month recurrence, catheterization time, length of hospital stay, postoperative bladder irrigation duration, obturator nerve reflex, bladder perforation, and no significant difference in the twelve-month recurrence, operation time, and urethral stricture compared with conventional transurethral resection for primary non-muscle invasive bladder cancer subjects. Further studies are required.

Vitamin D3 sensitizes resistant human bladder cancer cells to cisplatin by regulating Sirtuin 1 gene expression.

OBJECTIVE: Cisplatin is a standard chemotherapeutic agent for advanced bladder cancer, but its efficacy is limited due to drug resistance. Vitamin D3 may reverse cancer multidrug resistance, but the potential molecular mechanisms are still only partially known. The purpose of this study was to explore the mechanism by which vitamin D3 reverses cisplatin resistance in bladder cancer to improve therapeutic efficacy and ameliorate the prognosis of cisplatin-resistant bladder cancer. PATIENTS AND METHODS: The levels of vitamin D3 and sirtuin 1 protein were detected in cisplatin-resistant bladder cancer patients and cisplatin-sensitive patients. The cisplatin-resistant bladder cancer cell lines T24/DDP and UMUC3R were used as cell experimental models, and the migration, apoptosis, mitochondrial reactive oxygen species accumulation and autophagy of cells were assessed in the present study. RESULTS: Vitamin D3 levels were decreased, and sirtuin 1 protein levels were increased in cisplatin-resistant bladder cancer patients compared with cisplatin-sensitive bladder cancer patients. Vitamin D3 treatment markedly repressed sirtuin 1 expression, and overexpression of the sirtuin 1 gene led to mitochondrial reactive oxygen species generation, promoted the initiation of autophagosome formation and enhanced autophagic flux. Cisplatin treatment in the presence of vitamin D3 inhibited cell invasion and migration and induced apoptosis and enhancing the sirtuin 1 gene abolished the effect of vitamin D3 by regulating mitochondrial reactive oxygen species accumulation and autophagosome formation. CONCLUSIONS: These data support a mechanism wherein the sirtuin 1 gene plays a crucial role in vitamin D3 reversing cisplatin resistance in bladder cancer and may provide useful preventive and therapeutic applications in the future.

Antimicrobial Peptide Mechanism Studied by Scattering-Guided Molecular Dynamics Simulation.

In an effort to combat rising antimicrobial resistance, our labs have rationally designed cationic, helical, amphipathic antimicrobial peptides (AMPs) as alternatives to traditional antibiotics since AMPs incur bacterial resistance in weeks, rather than days. One highly positively charged AMP, WLBU2 (+13e), (RRWV RRVR RWVR RVVR VVRR WVRR), has been shown to be effective in killing both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria by directly perturbing the bacterial membrane nonspecifically. Previously, we used two equilibrium experimental methods: synchrotron X-ray diffuse scattering (XDS) providing lipid membrane thickness and neutron reflectometry (NR) providing WLBU2 depth of penetration into three lipid model membranes (LMMs). The purpose of the present study is to use the results from the scattering experiments to guide molecular dynamics (MD) simulations to investigate the detailed biophysics of the interactions of WLBU2 with LMMs of Gram-negative outer and inner membranes, and Gram-positive cell membranes, to elucidate the mechanisms of bacterial killing. Instead of coarse-graining, backmapping, or simulating without bias for several microseconds, all-atom (AA) simulations were guided by the experimental results and then equilibrated for ∼0.5 µs. Multiple replicas of the inserted peptide were run to probe stability and reach a combined time of at least 1.2 µs for G(-) and also 2.0 µs for G(+). The simulations with experimental comparisons help rule out certain structures and orientations and propose the most likely set of structures, orientations, and effects on the membrane. The simulations revealed that water, phosphates, and ions enter the hydrocarbon core when WLBU2 is positioned there. For an inserted peptide, the three types of amino acids, arginine, tryptophan, and valine (R, W, V), are arranged with the 13 Rs extending from the hydrocarbon core to the phosphate group, Ws are located at the interface, and Vs are more centrally located. For a surface state, R, W, and V are positioned relative to the bilayer interface as expected from their hydrophobicities, with Rs closest to the phosphate group, Ws close to the interface, and Vs in between. G(-) and G(+) LMMs are thinned ∼1 Å by the addition of WLBU2. Our results suggest a dual anchoring mechanism for WLBU2 both in the headgroup and in the hydrocarbon region that promotes a defect region where water and ions can flow across the slightly thinned bacterial cell membrane.

Rational Framework for the Design of Trp- and Arg-Rich Peptide Antibiotics Against Multidrug-Resistant Bacteria.

The threat of antibiotic resistance warrants the discovery of agents with novel antimicrobial mechanisms. Antimicrobial peptides (AMPs) directly disrupting bacterial membranes may overcome resistance to traditional antibiotics. AMP development for clinical use has been mostly limited to topical application to date. We developed a rational framework for systematically addressing this challenge using libraries composed of 86 novel Trp- and Arg-rich engineered peptides tested against clinical strains of the most common multidrug-resistant bacteria known as ESKAPE pathogens. Structure-function correlations revealed minimum lengths (as low as 16 residues) and Trp positioning for maximum antibacterial potency with mean minimum inhibitory concentration (MIC) of 2-4 µM and corresponding negligible toxicity to mammalian cells. Twelve peptides were selected based on broad-spectrum activity against both gram-negative and -positive bacteria and <25% toxicity to mammalian cells at maximum test concentrations. Most of the selected PAX remained active against the colistin-resistant clinical strains. Of the selected peptides, the shortest (the 16-residue E35) was further investigated for antibacterial mechanism and proof-of-concept in vivo efficacy. E35 killed an extensively-resistant isolate of Pseudomonas aeruginosa (PA239 from the CDC, also resistant to colistin) by irreversibly disrupting the cell membranes as shown by propidium iodide incorporation, using flow cytometry and live cell imaging. As proof of concept, in vivo toxicity studies showed that mice tolerated a systemic dose of up to 30 mg/kg peptide and were protected with a single 5 mg/kg intravenous (IV) dose against an otherwise lethal intraperitoneal injection of PA239. Efficacy was also demonstrated in an immune-compromised Klebsiella pneumoniae infection model using a daily dose of 4mg/kg E35 systemically for 2 days. This framework defines the determinants of efficacy of helical AMPs composed of only cationic and hydrophobic amino acids and provides a path for a potential departure from the restriction to topical use of AMPs toward systemic application.

Heterogeneous-Backbone Proteomimetic Analogues of Lasiocepsin, a Disulfide-Rich Antimicrobial Peptide with a Compact Tertiary Fold.

The emergence of resistance to clinically used antibiotics by bacteria presents a significant problem in public health. Natural antimicrobial peptides (AMPs) are a valuable source of antibiotics that act by a mechanism less prone to the evolutionary development of resistance. In an effort to realize the promise of AMPs while overcoming limitations such as poor biostability, researchers have sought sequence-defined oligomers with artificial amide-based backbones that show membrane-disrupting functions similar to natural agents. Most of this precedent has focused on short peptidomimetic analogues of unstructured chains or secondary folds; however, the natural antimicrobial arsenal includes a number of small- and medium-sized proteins that act via an ordered tertiary structure. Generating proteomimetic analogues of these scaffolds poses a challenge due to the increased complexity of the target for mimicry. Here, we report the development of heterogeneous-backbone variants of lasiocepsin, a 27-residue disulfide-rich AMP found in bee venom that adopts a compact tertiary fold. Iterative cycles of design, synthesis, and biological evaluation yielded analogues of the natural domain with ∼30 to 40% artificial backbone content, comparable antibacterial activity, reduced host cell toxicity, and improved stability to proteolytic degradation. High-resolution structures determined for several variants by NMR provide insights into the interplay among backbone composition, tertiary fold, and biological properties. Collectively, the results reported here broaden the scope of protein functional mimicry by artificial backbone analogues of tertiary folding patterns and suggest protein backbone engineering as a means to tune protein function by exerting site-specific control over protein folded structure.

Acidic Microenvironment Determines Antibiotic Susceptibility and Biofilm Formation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the most prevalent bacterial species that contribute to cystic fibrosis (CF) respiratory failure. The impaired function of CF transmembrane conductance regulator leads to abnormal epithelial Cl-/HCO3 - transport and acidification of airway surface liquid. However, it remains unclear why the CF lung is most commonly infected by Pseudomonas aeruginosa versus other pathogens. We carried out studies to investigate if lower pH helps Pseudomonas aeruginosa adapt and thrive in the CF-like acidic lung environment. Our results revealed that Pseudomonas aeruginosa generally forms more biofilm, induces antibiotic resistance faster in acidic conditions, and can be reversed by returning the acidic environment to physiologically neutral conditions. Pseudomonas aeruginosa appears to be highly adaptive to the CF-like acidic pH environment. By studying the effects of an acidic environment on bacterial response, we may provide a new therapeutic option in preventing chronic Pseudomonas aeruginosa infection and colonization.

Antimicrobial and Anti-Inflammatory Activity of Apple Polyphenol Phloretin on Respiratory Pathogens Associated With Chronic Obstructive Pulmonary Disease.

Bacterial infections contribute to accelerated progression and severity of chronic obstructive pulmonary disease (COPD). Apples have been associated with reduced symptoms of COPD and disease development due to their polyphenolic content. We examined if phloretin, an apple polyphenol, could inhibit bacterial growth and inflammation induced by the main pathogens associated with COPD. Phloretin displayed bacteriostatic and anti-biofilm activity against nontypeable Haemophilus influenzae (NTHi), Moraxella catarrhalis, Streptococcus pneumoniae, and to a lesser extent, Pseudomonas aeruginosa. In vitro, phloretin inhibited NTHi adherence to NCI-H292 cells, a respiratory epithelial cell line. Phloretin also exhibited anti-inflammatory activity in COPD pathogen-induced RAW 264.7 macrophages and human bronchial epithelial cells derived from normal and COPD diseased lungs. In mice, NTHi bacterial load and chemokine (C-X-C motif) ligand 1 (CXCL1), a neutrophil chemoattractant, was attenuated by a diet supplemented with phloretin. Our data suggests that phloretin is a promising antimicrobial and anti-inflammatory nutraceutical for reducing bacterial-induced injury in COPD.

Understanding the Host in the Management of Pneumonia. An Official American Thoracic Society Workshop Report.

Pneumonia causes a significant burden of disease worldwide. Although all populations are at risk of pneumonia, those at extremes of age and those with immunosuppressive disorders, underlying respiratory disease, and critical illness are particularly vulnerable. Although clinical practice guidelines addressing the management and treatment of pneumonia exist, few of the supporting studies focus on the crucial contributions of the host in pneumonia pathogenesis and recovery. Such essential considerations include the host risk factors that lead to susceptibility to lung infections; biomarkers reflecting the host response and the means to pursue host-directed pneumonia therapy; systemic effects of pneumonia on the host; and long-term health outcomes after pneumonia. To address these gaps, the Pneumonia Working Group of the Assembly on Pulmonary Infection and Tuberculosis led a workshop held at the American Thoracic Society meeting in May 2018 with overarching objectives to foster attention, stimulate research, and promote funding for short-term and long-term investigations into the host contributions to pneumonia. The workshop involved participants from various disciplines with expertise in lung infection, pneumonia, sepsis, immunocompromised patients, translational biology, data science, genomics, systems biology, and clinical trials. This workshop report summarizes the presentations and discussions and important recommendations for future clinical pneumonia studies. These recommendations include establishing consensus disease and outcome definitions, improved phenotyping, development of clinical study networks, standardized data and biospecimen collection and protocols, and development of innovative trial designs.

Pulmonary Inflammation and KRAS Mutation in Lung Cancer.

Chronic lung infection and lung cancer are two of the most important pulmonary diseases. Respiratory infection and its associated inflammation have been increasingly investigated for their role in increasing the risk of respiratory diseases including chronic obstructive pulmonary disease (COPD) and lung cancer. Kirsten rat sarcoma viral oncogene (KRAS) is one of the most important regulators of cell proliferation, differentiation, and survival. KRAS mutations are among the most common drivers of cancer. Lung cancer harboring KRAS mutations accounted for ~25% of the incidence but the relationship between KRAS mutation and inflammation remains unclear. In this chapter, we will describe the roles of KRAS mutation in lung cancer and how elevated inflammatory responses may increase KRAS mutation rate and create a vicious cycle of chronic inflammation and KRAS mutation that likely results in persistent potentiation for KRAS-associated lung tumorigenesis. We will discuss in this chapter regarding the studies of KRAS gene mutations in specimens from lung cancer patients and in animal models for investigating the role of inflammation in increasing the risk of lung tumorigenesis driven primarily by oncogenic KRAS.

Effects of blood pathological changes before TAI on pregnancy of dairy cows with anestrus and estrus / [Efeitos de mudanças patológicas de plasma antes de (TAI) na gestação de vacas em anestro e estro]

The objective of this study was to investigate the influence of plasma pathological changes before timed artificial insemination (TAI) on pregnancy of cows. The contents of estrogen (E2), progesterone (P4), glucose (Glu), selenium (Se), brain-derived neurotrophic factor (BDNF), and histamine (HIS) in plasma of 48 Holstein cows were measured before TAI. According to the estrus detection, the cows were divided into estrus (E) and anestrus (A) groups. After pregnancy testing at 28 d after TAI, two groups of E and A were divided into positive pregnancy of E group (EP+), negative pregnancy of E group (EP-), positive pregnancy of A group (AP+), and negative pregnancy of A group (AP-). The contents of E2, P4, Glu, Se, BDNF and hIS significantly differed among the four groups (P<0.01). The ROC analysis was used to determine the risk of negative pregnancy test (-) after TAI was increased when plasma E2 was less than 46.45 pmol/L in cows before TAI. The changes in E2, P4,hIS, Glu, and BDNF in the blood of natural estrus and natural anestrus cows affected the pregnancy after TAI. the level of E2 in plasma may be used to assess the risk of negative pregnancy after TAI.(AU)

O objetivo do presente estudo foi investigar a influência de mudanças patológicas de plasma antes de inseminação artificial (TAI) na gestação de vacas. O conteúdo de estrogênio (E2), progesterona (P4), glucose (Glu), selênio (Se), fator neurotrófico derivado do cérebro (BDNF), e histamina (HIS) no plasma de 48 vacas Holstein foi medido antes de TAI. De acordo com a detecção de estro, as vacas foram divididas em dois grupos: estro (E) e anestro (A). Após teste de gestação 28 d após TAI, dois grupos de E e A foram formados em gestação positiva do grupo E (EP+), gestação negativa do grupo E (EP-), gestação positiva do grupo A (AP+), e gestação negativa do grupo A (AP-). Os valores de E2, P4, Glu, Se, BDNF e hIS foram significativamente diferentes entre os quatro grupos (P<0,01). A análise ROC foi utilizada para determinar o risco de teste de gestação negativo (-) após aumento de TAI quando plasma E2 estava abaixo de 46,45 pmol/L em vacas antes de TAI. Alterações em E2, P4,hIS, Glu e BDNF no sangue de estro natural e anestro natural em vacas afetou a gestação após TAI. O nível de E2 no plasma pode ser usado para avaliar o risco de gestação negativa após TAI.(AU)

Genetic determinants of ammonia-induced acute lung injury in mice.

In this study, a genetically diverse panel of 43 mouse strains was exposed to ammonia, and genome-wide association mapping was performed employing a single-nucleotide polymorphism (SNP) assembly. Transcriptomic analysis was used to help resolve the genetic determinants of ammonia-induced acute lung injury. The encoded proteins were prioritized based on molecular function, nonsynonymous SNP within a functional domain or SNP within the promoter region that altered expression. This integrative functional approach revealed 14 candidate genes that included Aatf, Avil, Cep162, Hrh4, Lama3, Plcb4, and Ube2cbp, which had significant SNP associations, and Aff1, Bcar3, Cntn4, Kcnq5, Prdm10, Ptcd3, and Snx19, which had suggestive SNP associations. Of these genes, Bcar3, Cep162, Hrh4, Kcnq5, and Lama3 are particularly noteworthy and had pathophysiological roles that could be associated with acute lung injury in several ways.

Toll interacting protein protects bronchial epithelial cells from bleomycin-induced apoptosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by altered epithelial cell phenotypes, which are associated with myofibroblast accumulation in the lung. Atypical alveolar epithelial cells in IPF express molecular markers of airway epithelium. Polymorphisms within and around Toll interacting protein (TOLLIP) are associated with the susceptibility to IPF and mortality. However, the functional role of TOLLIP in IPF is unknown. Using lung tissues from IPF and control subjects, we showed that expression of TOLLIP gene in the lung parenchyma is globally lower in IPF compared to controls. Lung cells expressing significant levels of TOLLIP include macrophages, alveolar type II, and basal cells. TOLLIP protein expression is lower in the parenchyma of IPF lungs but is expressed in the atypical epithelial cells of the distal fibrotic regions. Using overexpression and silencing approaches, we demonstrate that TOLLIP protects cells from bleomycin-induced apoptosis using primary bronchial epithelial cells and BEAS-2B cells. The protective effects are mediated by reducing mitochondrial reactive oxygen species (ROS) levels and upregulating autophagy. Therefore, global downregulation of the TOLLIP gene in IPF lungs may predispose injured lung epithelial cells to apoptosis and to the development of IPF.

Enhanced therapeutic index of an antimicrobial peptide in mice by increasing safety and activity against multidrug-resistant bacteria.

The rising prevalence of antibiotic resistance underscores the urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs) are potentially effective therapeutics that disrupt bacterial membranes regardless of resistance to traditional antibiotics. We have developed engineered cationic AMPs (eCAPs) with broad activity against multidrug-resistant (MDR) bacteria, but stability remains an important concern. Therefore, we sought to enhance the clinical utility of eCAP WLBU2 in biological matrices relevant to respiratory infection. A designed substitution of d-Val for l-Val resulted in increased resistance to protease enzymatic degradation. We observed multiple gains of functions such as higher activity against bacteria in biofilm mode of growth, significantly lower toxicity to erythrocytes and white blood cells compared to WLBU2, with increased safety in mice. Direct airway delivery revealed a therapeutic index of >140 for the selected enantiomer compared to that of <35 for WLBU2. The data warrant clinical exploration by aerosolized delivery to mitigate MDR-related respiratory infection.

[Retinal toxicity study of intravitreal ganciclovir in albino rabbit].

Objective: To elevated the retinal toxicity of intravitreal ganciclovir in albino rabbit eyes. Methods: Experimental study. Twenty-four New Zealand albino rabbits (forty-eight eyes) were divided into four groups by random. Three groups were prepared for ganciclovir experiment, named A, B, C. Each group received intravitreal injection ganciclovir dose at 400 µg/0.05 ml, 2 mg/0.05 ml and 5 mg/0.05 ml respectively. The other group named D served as a control accepted intravitreal injection 0.9% normal saline 0.1 ml. Before and after 1, 2 and 4 weeks, flicker full field electroretina gram (ERG) was recorded. After 1, 2 and 4 weeks light and electron microscopic tests were recorded for further toxicity study. Results: There was significant difference in amplitude of maximal combined response a wave in one week(χ(2)=8.319, P=0.04), and pairwise comparison the 5 mg group (140.50 µV) was significantly lower than the control group (165.00 µV) (χ(2)=-2.830, P=0.028). Maximal combined response b wave in four weeks(χ(2)=-10.626, P=0.014), and pairwise comparison the 5 mg group (261.50 µV) was significantly lower than the control group (398.00 µV) (χ(2)=-2.973, P=0.018). 30 Hz flicker response in one, two and four weeks(χ(2)=17.589, 8.225, 8.997, P=0.001, 0.042, 0.02), and pairwise comparison the 5 mg group (71.3µV, 106.00µV, 63.60µV) was significantly lower than the control group (118.50µV, 129.00µV, 116.50µV) (χ(2)=-4.142, -2.826, -2.713, P=0.000, 0.028, 0.040). There was no histologic retinal toxicity evidence of group 400 µg and control group observed by light microscopy in any stage of the study. Histologic changes of group 2 mg four week later, group 5 mg two and four week later include inner nuclear layer loose arranged, nuclear of ganglia were widened and outer plexiform layer stained less in four week later. By electron microscopic observation, the ultrastructure of retina changed to different degrees and became worse in each experimental group with significant mitochondrial swelling and hydropic changes were seen in the inner segments of photoreceptors, loosely arranged and disordered in the outer segment of photoreceptors four weeks later. Conclusions: The retinal function and morphology were normal in group 400 µg. Group 2 mg and 5 mg had retinal toxicity, and 5 mg was more severe. Therefore, the clinical application of ganciclovir in the treatment of acute retinal necrosis (ARN) should select the minimum effective dose to avoid the occurrence of retinal toxicity. (Chin J Ophthalmol, 2020, 56:279-285).

Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes.

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.

Analysis of RNA Sequencing Data Using CLC Genomics Workbench.

RNA sequencing (RNA-seq) is a recently developed approach to perform transcriptome profiling using next-generation sequencing (NGS) technologies. Studies have shown that RNA-seq provides accurate measurement of transcript levels as well as their isoforms, which is useful to address complex transcriptomes. In addition, the increasing publicly available sequencing datasets and decreasing sequencing cost promote the use of RNA-seq for hypothesis-generating studies. In this chapter, we demonstrate how to analyze RNA-seq data and generate interpretable results using CLC genomic workbench software and perform the downstream pathway analysis using ingenuity pathway analysis (IPA).

Using Bronchoalveolar Lavage to Evaluate Changes in Pulmonary Diseases.

Bronchoalveolar lavage (BAL) is a procedure that can be used to collect samples from human and animal lungs to efficiently evaluate the immune response and the potentially pathological changes by examining both the compositions of cells and fluid from lavage. There are observable changes including inflammatory response in human and animal lungs exposed to environmental exposures such as toxic chemicals and microorganisms, or under pathophysiological conditions in respiratory system. The profile of inflammatory cells in BAL provides a qualitative description of inflammatory response, and the secretion in BAL fluid contains secreted proteins of inflammatory mediators and albumin as a quantitative measurement of inflammation and tissue injury in the lungs. Mouse is the most common model system being used for pulmonary disease-related research. A consistent experimental approach on how to lavage mouse lungs and collect samples from mouse lungs is important for a reproducible evaluation of pathological and physiological changes in mouse lung especially for the analysis of inflammation.

Fast and Efficient Measurement of Clinical and Biological Samples Using Immunoassay-Based Multiplexing Systems.

Immunoassay is one of the most commonly used biomedical techniques to detect the expression of an antibody or an antigen in a test sample. Enzyme-linked immunosorbent assay (ELISA) has been used for a variety of applications including diagnostic tools and quality controls. However, one of the main limitations of ELISA is its lack of multiplexing ability, so ELISA may not be an efficient diagnostic tool when a measurement of multiple determinants is needed for samples with limited quantity such as blood or biological samples from newborns or babies. Although similar to ELISA in assay measurement, multiplex platforms such as bead-based Luminex and multi-array-based MSD (Meso Scale Discovery) are widely used to measure multiple biomarkers from a single analysis. Luminex is a xMAP-based technology that combines several different technologies to provide an efficient and accurate measurement of multiple analytes from a single sample. The multiplexing can be achieved because up to 100 distinct Luminex color-coded microsphere bead sets can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. Using Multi-array and electrochemiluminescence technologies, the MSD platform provides the multiplex capability with similar consistence as observed in ELISA. Various biological samples that can be analyzed by both Luminex and MSD systems include serum, plasma, tissue and cell lysate, saliva, sputum, and bronchoalveolar Lavage (BAL). The most common Luminex and MSD-based assays are to detect a combined set of cytokines to provide a measurement of cytokine expression profiling for a diagnostic purpose.

Determination and Quantification of Bacterial Virulent Gene Expression Using Quantitative Real-Time PCR.

Polymerase chain reaction (PCR) plays significant roles in modern molecular biology. However, it is relatively cumbersome and less accurate to use the traditional PCR method in quantifying gene expression because it requires first generating a standard curve with multiple input controls showing linearity with amplified control PCR products on a electrophoresis gel to compare with the abundance of the to-be-determined gene transcript PCR amplicons. Quantitative real-time PCR (qRT-PCR) is a time-efficient and reliable tool for accurate quantification and comparison of gene (RNA transcript) expression from various biological samples. Current technology has simplified and expedited the qPCR process significantly. However, proper techniques and standard protocols are required in eliminating potentially erroneous experimental outcome. Here, we provide an example from a drug-treated bacterial gene expression study with detailed protocols to demonstrate real-time qPCR with SYBR™ Green and TaqMan®, two of the most adapted and well-established qPCR technologies. Relative quantification of gene (RNA transcript) expression using qRT-PCR is demonstrated in detail from sample preparations to data analysis.

A Rare Mutation in SPLUNC1 Affects Bacterial Adherence and Invasion in Meningococcal Disease.

BACKGROUND: Neisseria meningitidis (Nm) is a nasopharyngeal commensal carried by healthy individuals. However, invasive infections occurs in a minority of individuals, with devastating consequences. There is evidence that common polymorphisms are associated with invasive meningococcal disease (IMD), but the contributions of rare variants other than those in the complement system have not been determined. METHODS: We identified familial cases of IMD in the UK meningococcal disease study and the European Union Life-Threatening Infectious Disease Study. Candidate genetic variants were identified by whole-exome sequencing of 2 patients with familial IMD. Candidate variants were further validated by in vitro assays. RESULTS: Exomes of 2 siblings with IMD identified a novel heterozygous missense mutation in BPIFA1/SPLUNC1. Sequencing of 186 other nonfamilial cases identified another unrelated IMD patient with the same mutation. SPLUNC1 is an innate immune defense protein expressed in the nasopharyngeal epithelia; however, its role in invasive infections is unknown. In vitro assays demonstrated that recombinant SPLUNC1 protein inhibits biofilm formation by Nm, and impedes Nm adhesion and invasion of human airway cells. The dominant negative mutant recombinant SPLUNC1 (p.G22E) showed reduced antibiofilm activity, increased meningococcal adhesion, and increased invasion of cells, compared with wild-type SPLUNC1. CONCLUSIONS: A mutation in SPLUNC1 affecting mucosal attachment, biofilm formation, and invasion of mucosal epithelial cells is a new genetic cause of meningococcal disease.