Starting out rural: a qualitative study of the experiences of family physician graduates transitioning to practice in rural Ontario.

BACKGROUND: New family medicine graduates are a promising group to recruit to underserved rural areas. This study aimed to understand the experiences of this group as they transitioned to practice in rural Ontario. METHODS: We used a hermeneutic phenomenology approach. Purposive sampling was used to recruit participants who graduated from a Canadian family medicine residency program and worked in a rural community in Ontario (Rurality Index for Ontario score ≥ 40) for at least 1 year within the past 5 years. Participants completed an online demographic survey followed by a virtual semistructured interview (May-August 2022). Interviews were video recorded and transcribed. Two researchers reviewed transcripts for codes, and then codes were reviewed in an iterative process to create themes. Transcripts, codes and themes were reviewed by an independent researcher, and final themes were shared with participants to ensure reliability. RESULTS: We included 18 family physicians in the study. We identified 8 themes and 18 subthemes. The themes identified as important to the experience of new graduates were as follows: choosing rural practice, preparedness for practice, navigating work-life balance, navigating transition to practice, challenges during transition to practice, successes during transition to practice, locuming and emergency medicine as part of rural generalist practice. INTERPRETATION: Most physicians interviewed felt prepared for rural practice and enjoyed their work; however, they faced unique challenges associated with being an early-career physician in rural practice. This study identifies opportunities for improvements, which can guide medical educators, rural communities and their recruiters, new graduates and policy-makers.

Evaluation and Management of Iron Deficiency in Children Undergoing Intestinal Rehabilitation-A Position Paper From the NASPGHAN Intestinal Rehabilitation Special Interest Group.

Iron deficiency (ID) is the most common nutritional deficiency affecting children undergoing intestinal rehabilitation (IR). Patients may be asymptomatic or present with nonspecific symptoms including fatigue, irritability, and dizziness. The diagnosis of ID in this population can be complicated by the coexistence of systemic inflammation or other nutritional deficiencies which may mimic ID. Many routinely available laboratory tests lack specificity and no consensus on screening is available. Success in oral and enteral treatment is impeded by poor tolerance of iron formulations in a population already challenged with intolerance. Newer parenteral iron formulations exhibit excellent safety profiles, but their role in repletion in this population remains unclear. The following report, compiled by a multidisciplinary group of providers caring for children undergoing IR and representing the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Special Interest Group for Intestinal Rehabilitation, seeks to address these challenges. After discussing iron physiology and population-specific pathophysiology, we make recommendations on iron intake, iron status assessment, and evaluation for alternative causes of anemia. We then provide recommendations on iron supplementation and treatment of ID anemia specific to this nutritionally vulnerable population.

Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells.

INTRODUCTION: The taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance. METHODS: MCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR). RESULTS: MCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways. CONCLUSIONS: We report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance.

The effect of ABCB1 genetic variants on chemotherapy response in HIV and cancer treatment.

Despite their clearly distinct pathophysiologies, HIV and cancer are diseases whose response to chemotherapy treatment varies substantially amongst patients, in particular for those with prior drug exposure. This has been attributed, in part, to elevated expression of the ABCB1 drug transporter in some patients, which results in reduced drug accumulation in target tissues. Many mechanisms have been identified for this elevated expression of ABCB1, including variations in the sequence of the gene coding for the transporter (ABCB1). Over 50 SNPs within ABCB1 have been identified. Associations have been made between the presence of specific ABCB1 SNPs/haplotypes and both ABCB1 expression and the efficacy or toxicity of certain chemotherapy regimens. If these associations are strong and reproducibly demonstrated, then this would greatly aid in the development of individualized therapy regimes for specific cancer or HIV patients, based on their ABCB1 genotypes. This article highlights the significant recent progress made in this direction, but cautions that the utility of ABCB1 gene variants as biomarkers of chemotherapy drug response remains unclear to date.

Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation.

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the "gold standard" for measuring DNA methylation. However, with the evolution of pyrosequencing as a tool to evaluate DNA methylation, the need arises to compare the relative efficiencies of the two techniques in measuring DNA methylation. We provide for the first time a direct assessment of BSP and pyrosequencing to detect and quantify hypomethylation, hypermethylation, and mixed methylation of the ABCB1 promoter in various drug-sensitive and drug-resistant MCF-7 breast cancer cell lines through head-to-head experimentation. Our findings indicate that although both methods can reliably detect increased, decreased, and mixed methylation of DNA, BSP appears to be more sensitive than pyrosequencing at detecting strong hypermethylation of DNA. However, we also observed greater variability in the methylation of CpG sites by BSP, possibly due to the additional bacterial cloning step required by BSP over pyrosequencing. BSP and pyrosequencing equally detected hypomethylation and mixed methylation of DNA. The ability of pyrosequencing to reliably detect differences in DNA methylation across cell populations without requiring the cloning of bisulfite-treated DNA into bacterial expression vectors was seen as a major advantage of this technique.

Hypermethylation of the ABCB1 downstream gene promoter accompanies ABCB1 gene amplification and increased expression in docetaxel-resistant MCF-7 breast tumor cells.

Drug transporters have been implicated in resistance of solid and non-solid tumors to a variety of chemotherapeutic agents. Higher expression of the ABCB1 drug transporter is often observed in drug-resistant tumor cells, although the precise mechanism remains unclear. During selection of MCF-7 cells for survival in increasing concentrations of docetaxel (MCF-7TXT cells), we observed in this study a temporal correlation between the acquisition of docetaxel resistance at selection dose 9 and the increased expression of ABCB1. Both the magnitude of docetaxel resistance and the level of ABCB1 expression then rose as the selection dose was further elevated. We also observed through bisulfite sequencing experiments that the ABCB1 downstream promoter became increasingly methylated following the acquisition of drug resistance (selection doses 10-12). Transcription was solely attributed to the upstream ABCB1 promoter within MCF-7TXT cells at the highest selection dose suggesting that hypermethylation caused a shift in promoter usage. The hypermethylation was also accompanied by regional amplification of chromosome 7 containing the ABCB1 gene and its neighbor ABCB4 but not DBF-4. The amplification of the ABCB1 gene correlated positively both with the hypermethylation of the ABCB1 downstream promoter (r=0.90) and the increased expression of ABCB1 (r=0.78). Moreover demethylation of the ABCB1 downstream promoter induced by 5-aza-2A'deoxycytidine treatment decreased the expression of ABCB1 mRNA in MCF-7TXT cells. Taken together, our findings suggest that the increased expression of ABCB1 upon acquisition of docetaxel resistance in breast tumor cells can be multifactorial, involving both epigenetic changes in promoter usage and regional chromosome amplification.

Scanning mutagenesis studies reveal multiple distinct regions within the human protein kinase C alpha regulatory domain important for phorbol ester-dependent activation of the enzyme.

While phorbol ester-binding sites within protein kinase C alpha (PKCalpha) have been identified and characterized utilizing fragments of the enzyme, it remains unclear whether additional regions within the enzyme may play an important role in its ability to be activated by phorbol ester. To examine this hypothesis, we generated 20 glutathione-S-transferase-tagged, V1-deficient, human PKCalpha holoenzyme constructs in which tandem six or 12 amino acid residue stretches along the full regulatory domain were changed to alanine residues. Each protein was assessed for its ability to bind phorbol ester and to induce growth repression when its catalytic activity was activated by phorbol ester upon expression in yeast cells. Mutagenesis of residues 99-158 potently reduced phorbol binding, consistent with previously published findings on the importance of the C1b region in phorbol binding. In addition, we identified a number of regions within the PKC regulatory domain that, when mutagenized, blocked the activation of PKC-mediated growth repression by phorbol ester while actually enhancing phorbol ester binding in vitro (residues 33-62, and 75-86). This study thus helps distinguish regions important for phorbol binding from regions important for the ability of phorbol ester to activate the enzyme. Our findings also suggest that multiple regions within C2 are necessary for full activation of the enzyme by phorbol ester, in particular residues 231-254. Finally, three regions, when mutagenized, completely, blocked catalytic domain activity in vivo (residues 33-62, 75-86, and 123-146), underscoring the important role of regulatory domain sequences in influencing catalytic domain function, even in the absence of the V1 region containing the pseudosubstrate sequence. This is the first tandem mutagenesis study for PKC that assesses the importance of regions for both phorbol binding and for phorbol-dependent activation in the context of the entire holoenzyme.