Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Agricultural biotechnology for crop improvement in a variable climate: hope or hype?. Review

Developing crops that are better adapted to abiotic stresses is important for food production in many parts of the world today. Anticipated changes in climate and its variability, particularly extreme temperatures and changes in rainfall, are expected to make crop improvement even more crucial for food production. Here, we review two key biotechnology approaches, molecular breeding and genetic engineering, and their integration with conventional breeding to develop crops that are more tolerant of abiotic stresses. In addition to a multidisciplinary approach, we also examine some constraints that need to be overcome to realize the full potential of agricultural biotechnology for sustainable crop production to meet the demands of a projected world population of nine billion in 2050

Engineered polyallylamine nanoparticles for efficient in vitro transfection

Purpose: Cationic polymers (i.e. polyallylamine, poly-L-lysine) having primary amino groups are poor transfection agents and possess high cytotoxicity index when used without any chemical modification and usually entail specific receptor mediated endocytosis or lysosomotropic agents to execute efficient gene delivery. In this report, primary amino groups of polyallylamine (PAA, 17 kDa) were substituted with imidazolyl functions, which are presumed to enhance endosomal release, and thus enhance its gene delivery efficiency and eliminate the requirement of external lysosomotropic agents. Further, systems were cross-linked with polyethylene glycol (PEG) to prepare PAA-IAA-PEG (PIP) nanoparticles and evaluated them in various model cell lines. Materials and Methods: The efficacy of PIP nanoparticles in delivering a plasmid encoding enhanced green fluorescent protein (EGFP) gene was assessed in COS-1, N2a and HEK293 cell lines, while their cytotoxicity was investigated in COS-1 and HEK293 cell lines. The PAA was chemically modified using imidazolyl moieties and ionically cross-linked with PEG to engineer nanoparticles. The extent of substitution was determined by ninhydrin method. The PIP nanoparticles were further characterized by measuring the particle size (dynamic light scattering and transmission electron microscopy), surface charge (zeta potential), DNA accessibility and buffering capacity. The cytotoxicity was examined using the MTT method. Results: In vitro transfection efficiency of synthesized nanoparticles is increased up to several folds compared to native polymer even in the presence of serum, while maintaining the cell viability over 100% in COS-1 cells. Nanoparticles possess positive zeta potential between 5.6-13 mV and size range of 185-230 nm in water. The accessibility experiment demonstrated that nanoparticles with higher degree of imidazolyl substitution formed relatively loose complexes with DNA. An acid-base titration showed enhanced buffering capacity of modified PAA. Conclusions: The PIP nanoparticles reveal tremendous potential as novel delivery system for achieving improved transfection efficiency, while keeping the cells at ease

Differences in multiple immune parameters between Indian and U.S. infants.

To compare immune phenotypes across two geographic and ethnic communities, we examined umbilical cord blood by flow cytometry and Luminex in parallel cohorts of 53 newborns from New Delhi, India, and 46 newborns from Stanford, California. We found that frequencies of a B cell subset suggested to be B-1-like, and serum IgM concentration were both significantly higher in the Stanford cohort, independent of differences in maternal age. While serum IgA levels were also significantly higher in the Stanford cohort, IgG1, IgG2, and IgG4 were significantly higher in the New Delhi samples. We found that neutrophils, plasmacytoid dendritic cells, CD8+ T cells, and total T cells were higher in the U.S. cohort, while dendritic cells, patrolling monocytes (CD14dimCD16+), natural killer cells, CD4+ T cells, and naïve B cells were higher in the India cohort. Within the India cohort, we also identified cell types whose frequency was positively or negatively predictive of occurrence of infection(s) in the first six months of life. Monocytes, total T cells, and memory CD4+ T cells were most prominent in having an inverse relationship with infection. We suggest that these data provide impetus for follow-up studies linking phenotypic differences to environmental versus genetic factors, and to infection outcomes

Comparison of Human Neonatal and Adult Blood Leukocyte Subset Composition Phenotypes.

The human peripheral leukocyte subset composition depends on genotype variation and pre-natal and post-natal environmental influence diversity. We quantified this composition in adults and neonates, and compared the median values and dispersal ranges of various subsets in them. We confirmed higher frequencies of monocytes and regulatory T cells (Tregs), similar frequencies of neutrophils, and lower frequencies of CD8 T cells, NKT cells, B1 B cells and gamma-delta T cells in neonatal umbilical cord blood. Unlike previous reports, we found higher frequencies of eosinophils and B cells, higher CD4:CD8 ratios, lower frequencies of T cells and iNKT cells, and similar frequencies of CD4 T cells and NK cells in neonates. We characterized monocyte subsets and dendritic cell (DC) subsets in far greater detail than previously reported, using recently described surface markers and gating strategies and observed that neonates had lower frequencies of patrolling monocytes and lower myeloid dendritic cell (mDC):plasmacytoid DC (pDC) ratios. Our data contribute to South Asian reference values for these parameters. We found that dispersal ranges differ between different leukocyte subsets, suggesting differential determination of variation. Further, some subsets were more dispersed in adults than in neonates suggesting influences of postnatal sources of variation, while some show the opposite pattern suggesting influences of developmental process variation. Together, these data and analyses provide interesting biological possibilities for future exploration

Underweight Full-Term Indian Neonates Show Differences in Umbilical Cord Blood Leukocyte Phenotype: A Cross-Sectional Study

<div><p>Background</p><p>While infections are a major cause of neonatal mortality in India even in full-term neonates, this is an especial problem in the large proportion (~20%) of neonates born underweight (or small-for-gestational-age; SGA). One potential contributory factor for this susceptibility is the possibility that immune system maturation may be affected along with intrauterine growth retardation.</p><p>Methods</p><p>In order to examine the possibility that differences in immune status may underlie the susceptibility of SGA neonates to infections, we enumerated the frequencies and concentrations of 22 leukocyte subset populations as well as IgM and IgA levels in umbilical cord blood from full-term SGA neonates and compared them with values from normal-weight (or appropriate-for-gestational-age; AGA) full-term neonates. We eliminated most SGA-associated risk factors in the exclusion criteria so as to ensure that AGA-SGA differences, if any, would be more likely to be associated with the underweight status itself.</p><p>Results</p><p>An analysis of 502 such samples, including 50 from SGA neonates, showed that SGA neonates have significantly fewer plasmacytoid dendritic cells (pDCs), a higher myeloid DC (mDC) to pDC ratio, more natural killer (NK) cells, and higher IgM levels in cord blood in comparison with AGA neonates. Other differences were also observed such as tendencies to lower CD4:CD8 ratios and greater prominence of inflammatory monocytes, mDCs and neutrophils, but while some of them had substantial differences, they did not quite reach the standard level of statistical significance.</p><p>Conclusions</p><p>These differences in cellular lineages of the immune system possibly reflect stress responses in utero associated with growth restriction. Increased susceptibility to infections may thus be linked to complex immune system dysregulation rather than simply retarded immune system maturation.</p></div

NK cells, but not innate-like T cells, show significant difference between AGA and SGA cord blood.

<p>(A) Comparison of proportions and absolute concentrations of NKT, iNKT and TCRγ/δ cells (median, IQR and 95% CI) along with p values. (B) Comparison of proportions and absolute numbers of immature NK cells (median, IQR and 95% CI) along with p values.</p

Characteristics of participant population.

<p>^<i>a</i> All values are Mean (SD) except where specified</p><p>^<i>b</i> To convert zinc in μg/dL to SI unit (μmol/L) multiply by 0.153</p><p>SGA, small for gestational age (birth weight below the 10th centile or 2SD below mean for GA of reference/normal birth curves); AGA, appropriate for gestational age (birth weight between the 10^th and 90^th centile for GA of reference/normal birth curves).</p><p>Characteristics of participant population.</p

Classical monocytes, myeloid and plasmacytoid DCs as well as mDC: pDC ratios differ between AGA and SGA cord blood.

<p>(A) Comparison of proportions of monocyte subsets and absolute concentrations (median, IQR and 95% CI) along with p values. (B) Comparison of proportions of DC subsets and absolute concentrations (median, IQR and 95% CI) along with p values. (C) Comparison of mDC: pDC ratios in cord blood (median, IQR and 95% CI) along with p value. (D) Comparison of concentrations of zinc in cord blood (median, IQR and 95% CI) along with p value.</p