An Investigation of the Types of Problems Faced by Small Firms and How They Affect the Funding Choices Made by Three Distinct Market Segments

This article looks at the relationship between the problems faced by small business owners and the funding sources used to solve those problems. Three problem types are identified: organizational systems, external, and sales and marketing problems. Based on these three problem types and the funding sources used by owners, the market is segmented into three groups using cluster analysis. Segment 1 is made up of firms with few problems. This segment uses the widest array of financial sources. Segment 2 has more problems than segment 3, but both need help with organizational systems resulting in the use of fewer sources

Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease

The US Distribution of Physicians from Lower Income Countries

Introduction: Since the 1960 s, the number of international medical graduates (IMGs) in the United States has increased significantly. Given concerns regarding the effects of this loss to their countries of origin, the authors undertook a study of IMGs from lower income countries currently practicing in the United States. Methods: The AMA Physician Masterfile was accessed to identify all 265,851 IMGs in active practice in the United States. These were divided by state of practice and country of origin. World Bank income classification was used to identify lowe

Peptide super-agonist enhances T-cell responses to melanoma

Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted “universal” cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV “magic bullet.

Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria

BACKGROUND: Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases. METHODOLOGY: This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion. SIGNIFICANCE: Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria

A SNAP-Tagged Derivative of HIV-1—A Versatile Tool to Study Virus-Cell Interactions

Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches

Efficient ex vivo expansion of conserved element vaccine-specific CD8+ T-cells from SHIV-infected, ART-suppressed nonhuman primates

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells’ recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo-expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells’ predominant CD8+ effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses

Differential Roles of HOW in Male and Female Drosophila Germline Differentiation

The adult gonads in both male and female Drosophila melanogaster produce gametes that originate from a regenerative pool of germline stem cells (GSCs). The differentiation programme that produces gametes must be co-ordinated with GSC maintenance and proliferation in order to regulate tissue regeneration. The HOW RNA-binding protein has been shown to maintain mitotic progression of male GSCs and their daughters by maintenance of Cyclin B expression as well as suppressing accumulation of the differentiation factor Bam. Loss of HOW function in the male germline results in loss of GSCs due to a delay in G2 and subsequent apoptosis. Here we show that female how mutant GSCs do not have any cell cycle defects although HOW continues to bind bam mRNA and suppress Bam expression. The role of HOW in suppressing germ cell Bam expression appears to be conserved between sexes, leading to different cellular outcomes in how mutants due to the different functions of Bam. In addition the role in maintaining Cyclin B expression has not been conserved so female how GSCs differentiate rather than arrest