Characterizing a panel of amino acid auxotrophs under auxotrophic starvation conditions.

Auxotrophic strains starving for their cognate nutrient, termed auxotrophic starvation, are characterized by a shorter lifespan, higher glucose wasting phenotype, and inability to accomplish cell cycle arrest when compared to a "natural starvation," where a cell is starving for natural environmental growth-limiting nutrients such as phosphate. Since evidence of this physiological response is limited to only a subset of auxotrophs, we evaluated a panel of auxotrophic mutants to determine whether these responses are characteristic of a broader range of amino acid auxotrophs. Based on the starvation survival kinetics, the panel of strains was grouped into three categories-short-lived strains, strains with survival similar to a prototrophic wild type strain, and long-lived strains. Among the short-lived strains, we observed that the tyrosine, asparagine, threonine, and aspartic acid auxotrophs rapidly decline in viability, with all strains unable to arrest cell cycle progression. The three basic amino acid auxotrophs had a survival similar to a prototrophic strain starving in minimal media. The leucine, tryptophan, methionine, and cysteine auxotrophs displayed the longest lifespan. We also demonstrate how the phenomenon of glucose wasting is limited to only a subset of the tested auxotrophs, namely the asparagine, leucine, and lysine auxotrophs. Furthermore, we observed pleiotropic phenotypes associated with a subgroup of auxotrophs, highlighting the importance of considering unintended phenotypic effects when using auxotrophic strains especially in chronological aging experiments.

Disorders of hyperpigmentation. Part II. Review of management and treatment options for hyperpigmentation.

Key challenges in the management of pigmentary disorders such as melasma and postinflammatory hyperpigmentation are their resistance to treatment, tendency to recur after treatment, and the risk of exacerbating hyperpigmentation with many treatment modalities. The second article in this 2-part continuing medical education series on pigmentary disorders focuses on the evidence behind medical and procedural treatments of dyschromias, including photoprotection, topical lightening agents, oral agents, chemical peels, and laser therapy.

Disorders of hyperpigmentation. Part I. Pathogenesis and clinical features of common pigmentary disorders.

Disorders of hyperpigmentation are common and, depending on the extent and location of involvement, can affect the quality of life and pose a significant psychologic burden for patients. Given the similarities in presentation of the various causes of hyperpigmentation, it is often difficult to elucidate the etiology of these conditions, which is important to guide management. Furthermore, certain disorders, such as lichen planus pigmentosus and ashy dermatosis, have similar clinical and/or histologic presentations, and their classification as distinct entities has been debated upon, leading to additional confusion. In this review, the authors selected commonly encountered disorders of hyperpigmentation of the skin, subdivided into epidermal, dermal, or mixed epidermal-dermal disorders based on the location of pigment deposition, along with disorders of hyperpigmentation of the mucosa and nails. Melanocytic nevi, genetic disorders, and systemic causes of hyperpigmentation were largely excluded and considered to be outside the scope of this review. We discussed the pathogenesis of hyperpigmentation as well as the clinical and histologic features of these conditions, along with challenges encountered in their diagnosis and classification. The second article in this 2-part continuing medical education series focuses on the medical and procedural treatments of hyperpigmentation.

A genome-scale yeast library with inducible expression of individual genes.

The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3 EB42, that gives lower expression than Z3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.

Preparing for colorectal surgery: a qualitative study of experiences and preferences of patients in Western Canada.

OBJECTIVES: The burden and costs of abdominal surgery for chronic conditions are on the rise, but could be reduced through self-management support. However, structured support to prepare for colorectal surgery is not routinely offered to patients in Canada. This study aimed to describe experiences and explore preferences for multimodal prehabilitation among colorectal surgery patients. METHODS: A qualitative descriptive study using three focus groups (FG) was held with 19 patients who had a surgical date for abdominal surgery (April 2017-April 2018) and lived close (≤ 50 km radius) to a tertiary hospital in Western Canada (including a Surgical Lead for the British Columbia Enhanced Recovery After Surgery (ERAS) Collaborative). FGs were audio-taped and verbatim transcribed with coding and pile-and-sort methods performed by two independent reviewers, confirmed by a third reviewer, in NVivo v9 software; followed by thematic analysis and narrative synthesis. RESULTS: Four themes emerged: support, informed decision-making, personalization of care, and mental/emotional health, which patients felt was particularly important but rarely addressed. Patient preferences for prehabilitation programming emphasised regular support from a single professional source, simple health messages, convenient access, and flexibility. CONCLUSIONS: There is an unmet need for structured preoperative support to better prepare patients for colorectal surgery. Future multimodal prehabilitation should be flexible and presented with non-medical information so patients can make informed decisions about their preoperative care and surgical outcomes. Healthcare providers have an important role in encouraging healthy lifestyle changes before colorectal surgery, though clearer communication and accurate advice on self-care, particularly mental health, are needed for improving patient outcomes.

Accelerating and de-risking CMC development with transposon-derived manufacturing cell lines.

The development of highly productive, genetically stable manufacturing cell lines is on the critical path to IND filing for protein-based biologic drugs. Here, we describe the Leap-In Transposase® platform, a novel transposon-based mammalian (e.g., Chinese hamster ovary) cell line development system that produces high-titer stable pools with productivity and product quality attributes that are highly comparable to clones that are subsequently derived therefrom. The productivity distributions of clones are strongly biased toward high producers, and genetic and expression stability is consistently high. By avoiding the poor integration rates, concatemer formation, detrimental transgene recombination, low average expression level, unpredictable product quality, and inconsistent genetic stability characteristic of nonhomologous recombination methods, Leap-In provides several opportunities to de-risk programs early and reduce timelines and resources.

Clinical prediction tool for extended-spectrum beta-lactamase-producing enterobacterales as the etiology of a bloodstream infection in solid organ transplant recipients.

BACKGROUND: Multidrug-resistant Gram-negative bacterial infections are increasingly common among solid organ transplant (SOT) recipients, leading to challenges in the selection of empiric antimicrobial therapy. We sought to develop a clinical tool to predict which SOT recipients are at high risk for extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (EB) bloodstream infection (BSI). METHODS: A multicenter case-control study was performed. The source population included SOT recipients with an EB BSI between 2005 and 2018. Cases were those with ESBL-EB BSI; controls were those with non-ESBL EB BSI. The population was subdivided into derivation and validation cohorts based on study site. The predictive tool was developed in the derivation cohort through iterative multivariable logistic regression analyses that maximized the area under the receiver-operating curve (AUC). External validity was assessed using the validation cohort. RESULTS: A total of 897 SOT recipients with an EB BSI were included, of which 539 were assigned to the derivation cohort (135, 25% ESBL-EB) and 358 to the validation cohort (221, 62% ESBL-EB). Using multivariable analyses, the most parsimonious model that was predictive of ESBL-EB BSI consisted of 10 variables, which fell into four clinical categories: prior colonization or infection with EB organisms, recent antimicrobial exposures, severity of preceding illness, and immunosuppressive regimen. This model achieved an AUC of 0.81 in the derivation cohort and 0.68 in the validation cohort. CONCLUSIONS: Though further refinements are needed in additional populations, this tool shows promise for guiding empiric therapy for SOT recipients with EB BSI.

Internal migration and health in South Africa: determinants of healthcare utilisation in a young adult cohort.

BACKGROUND: In South Africa, human geographic mobility is high as people engage in both permanent and temporary relocation, predominantly from rural to urban areas. Such mobility can compromise healthcare access and utilisation. The objective of this paper is to explore healthcare utilisation and its determinants in a cohort of internal migrants and permanent residents (non-migrants) originating from the Agincourt sub-district in South Africa's rural northeast. METHODS: A 5-year cohort study of 3800 individuals aged 18 to 40 commenced in 2017. Baseline data have been collected from 1764 Agincourt residents and 1334 temporary, mostly urban-based, migrants, and are analysed using bivariate analyses, logistic and multinomial regression models, and propensity score matching analysis. RESULTS: Health service utilisation differs sharply by migrant status and sex. Among those with a chronic condition, migrants had 0.33 times the odds of non-migrants to have consulted a health service in the preceding year, and males had 0.32 times the odds of females of having used health services. Of those who utilised services, migration status was further associated with the type of healthcare utilised, with 97% of non-migrant rural residents having accessed government facilities, while large proportions of migrants (31%) utilised private health services or consulted traditional healers (25%) in migrant destinations. The multinomial logistic regression analysis indicated that, in the presence of controls, migrants had 8.12 the relative risk of non-migrants for utilising private healthcare (versus the government-services-only reference category), and 2.40 the relative risk of non-migrants for using a combination of public and private sector facilities. These findings of differential utilisation hold under statistical adjustment for relevant controls and for underlying propensity to migrate. CONCLUSIONS: Migrants and non-migrants in the study population in South Africa were found to utilise health services differently, both in overall use and in the type of healthcare consulted. The study helps improve upon the limited stock of knowledge on how migrants interface with healthcare systems in low and middle-income country settings. Findings can assist in guiding policies and programmes to be directed more effectively to the populations most in need, and to drive locally adapted approaches to universal health coverage.

Improved patient access and outcomes with the integration of an eConsult program (teledermatology) within a large academic medical center.

BACKGROUND: Insurance, racial, and socioeconomic health disparities continue to pose significant challenges for access to dermatologic care. Studies applying teledermatology to increase access to underinsured individuals and ethnic minorities are limited. OBJECTIVE: To determine how the implementation of a teledermatology program affects access to health care and patient outcomes. METHODS: A cross-sectional evaluation was performed of all ambulatory dermatology referrals and electronic dermatology consultations (eConsults) at Ohio State University within a 25-month period. RESULTS: Compared with ambulatory referrals, eConsults served more nonwhite patients (612 of 1698 [36.0%] vs 4040 of 16,073 [25.1%]; P < .001) and more Medicaid enrollees (459 of 1698 patients [27.0%] vs 3266 of 16,073 [20.3%]; P < .001). In addition, ambulatory referral patients were significantly less likely to attend their scheduled appointment compared with eConsult patients, as either "no-shows" (246 of 2526 [9.7%] vs 3 of 62 [4.8%]) or cancellations (742 of 2526 [29.4%] vs 8 of 62 [12.9%]; P = .003). There were fewer median days to extirpation for eConsult patients compared with ambulatory referral patients (interquartile range; 80.7 ± 79.8 vs 116.9 ± 86.6 days; P = .004). CONCLUSION: Integrating dermatologic care through a telemedicine system can result in improved access for underserved patients through improved efficiency outcomes.

Lipid transporter Spns2 promotes microglia pro-inflammatory activation in response to amyloid-beta peptide.

Accumulating evidence indicates that neuroinflammation contributes to the pathogenesis and exacerbation of neurodegenerative disorders, such as Alzheimer's disease (AD). Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid that regulates many pathophysiological processes including inflammation. We present evidence here that the spinster homolog 2 (Spns2), a S1P transporter, promotes microglia pro-inflammatory activation in vitro and in vivo. Spns2 knockout (Spns2KO) in primary cultured microglia resulted in significantly reduced levels of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) and amyloid-beta peptide 1-42 oligomers (Aß42) when compared with littermate controls. Fingolimod (FTY720), a S1P receptor 1 (S1PR1) functional antagonist and FDA approved drug for relapsing-remitting multiple sclerosis, partially blunted Aß42-induced pro-inflammatory cytokine generation, suggesting that Spns2 promotes microglia pro-inflammatory activation through S1P-signaling. Spns2KO significantly reduced Aß42-induced nuclear factor kappa B (NFκB) activity. S1P increased, while FTY720 dampened, Aß42-induced NFκB activity, suggesting that Spns2 activates microglia inflammation through, at least partially, NFκB pathway. Spns2KO mouse brains showed significantly reduced Aß42-induced microglia activation/accumulation and reduced levels of pro-inflammatory cytokines when compared with age-matched controls. More interestingly, Spns2KO ameliorated Aß42-induced working memory deficit detected by Y-Maze. In summary, these results suggest that Spns2 promotes pro-inflammatory polarization of microglia and may play a crucial role in AD pathogenesis.

Unraveling incontinentia pigmenti: A comparison of phenotype and genotype variants.

BACKGROUND: Incontinentia pigmenti (IP) is a rare X-linked dominant genodermatosis that affects multiple systems with highly variable phenotypic expressivity. Although most affected individuals carry a common pathogenic variant on the IKBKG gene, approximately 20% have no identifiable mutation. OBJECTIVE: To describe clinical characteristics and genotype of IP patients and compare clinical differences between IKBKG pathogenic variant positive and negative cohorts. METHODS: Retrospective cohort study conducted at a large tertiary pediatric center from 1990 to 2017, for children with a clinical diagnosis of IP. RESULTS: Forty-two children with IP were identified, including 33 of 42 (79%) females. Most presented with cutaneous stage I findings (31 of 42; 74%). Extracutaneous involvements were common: dental (50%), ocular (31%), hair (31%), nail (15%), and neurodevelopmental (26%). An IKBKG pathogenic variant was detected in 20 of 34 (59%) patients. Compared with these, 14 of 34 (41%) patients who tested negative were significantly more likely (P < .05) to be male, have no family history of IP, and have lower incidences of dental and hair anomalies. LIMITATIONS: Retrospective methodology limits clear determination of the temporality of symptoms. CONCLUSION: Clinical differences between IKBKG pathogenic variant positive and negative IP cohorts support the prognostic utility of molecular genetic evaluation.

Folliculotropic mycosis fungoides in a pediatric patient mimicking black dot tinea capitis.

Immunosuppression following organ transplantation is a known risk factor for the development of lymphoproliferative disorders. Mycosis fungoides, a rare entity in pediatric patients, has seldom been reported as a post-transplant lymphoproliferative disorder. We report a case of folliculotropic mycosis fungoides in a pediatric patient following liver transplantation that was initially diagnosed as tinea capitis.

The Effectiveness of an International Cleft Mission Model in Asia: An Update.

BACKGROUND: Cleft lip/palate is a congenital craniofacial anomaly affecting patients physically and psychosocially and has contributed to the global burden of surgical disease, especially in underprivileged areas. For 20 years, Noordhoff Craniofacial Foundation (NCF) and the Chang Gung Craniofacial Center (CGCFC) have carried out missions to these areas. Rather than implementing short-term missions that lack proper follow-up care, the team has provided an effective, long-term, and multidisciplinary approach for the treatment of patients with cleft lip/palate. In this study, we evaluate the sustainability and effectiveness of the cleft mission model implemented by NCF and CGCFC. METHODS: Data from the years 1998-2017 were retrieved from the NCF database. All local centers were evaluated by a 3-stage categorization, levels 1 to 3, based on 4 criteria: (1) capacity to carry out independent missions, (2) diversity of cleft-care professionals, (3) diversity of surgical service offered, and (4) collaboration with local hospitals. Support and training of personnel were provided based on deficiency in these criteria. Noordhoff Craniofacial Foundation made close collaborations and partnerships with several organizations that shared its mission for comprehensive cleft care in developing countries. RESULTS: In all, 19 partner cleft teams in 9 different countries were established. In coordination with these teams, NCF and CGCFC have treated 1846 patients across 78 mission trips. To date, 158 personnel from 19 different countries have been successfully trained to provide cleft care in local centers. Most partner cleft teams centers have progressively reached category level 3, including those in the Philippines, Cambodia, and Mongolia. CONCLUSIONS: In order to establish and maintain sustainable cleft care in developing regions, commitment and compassion toward those who lack essential resources are necessary. Noordhoff Craniofacial Foundation and CGCFC have achieved a successful and practicable model through seeding medical personnel in order to provide effective and sustainable cleft care to the regions in need.

Increased liver tumor formation in neutral sphingomyelinase-2-deficient mice.

Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C16-ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplets, suggesting that nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs) and higher levels of phosphorylated signal transducer and activator of transcription 3, an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs, leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis.

Long term perfusion system supporting adipogenesis.

Adipose tissue engineered models are needed to enhance our understanding of disease mechanisms and for soft tissue regenerative strategies. Perfusion systems generate more physiologically relevant and sustainable adipose tissue models, however adipocytes have unique properties that make culturing them in a perfusion environment challenging. In this paper we describe the methods involved in the development of two perfusion culture systems (2D and 3D) to test their applicability for long term in vitro adipogenic cultures. It was hypothesized that a silk protein biomaterial scaffold would provide a 3D framework, in combination with perfusion flow, to generate a more physiologically relevant sustainable adipose tissue engineered model than 2D cell culture. Consistent with other studies evaluating 2D and 3D culture systems for adipogenesis we found that both systems successfully model adipogenesis, however 3D culture systems were more robust, providing the mechanical structure required to contain the large, fragile adipocytes that were lost in 2D perfused culture systems. 3D perfusion also stimulated greater lipogenesis and lipolysis and resulted in decreased secretion of LDH compared to 2D perfusion. Regardless of culture configuration (2D or 3D) greater glycerol was secreted with the increased nutritional supply provided by perfusion of fresh media. These results are promising for adipose tissue engineering applications including long term cultures for studying disease mechanisms and regenerative approaches, where both acute (days to weeks) and chronic (weeks to months) cultivation are critical for useful insight.

Epidermal growth factor receptor plays a role in the regulation of liver and plasma lipid levels in adult male mice.

Dsk5 mice have a gain of function in the epidermal growth factor receptor (EGFR), caused by a point mutation in the kinase domain. We analyzed the effect of this mutation on liver size, histology, and composition. We found that the livers of 12-wk-old male Dsk5 heterozygotes (+/Dsk5) were 62% heavier compared with those of wild-type controls (+/+). The livers of the +/Dsk5 mice compared with +/+ mice had larger hepatocytes with prominent, polyploid nuclei and showed modestly increased cell proliferation indices in both hepatocytes and nonparenchymal cells. An analysis of total protein, DNA, and RNA (expressed relative to liver weight) revealed no differences between the mutant and wild-type mice. However, the livers of the +/Dsk5 mice had more cholesterol but less phospholipid and fatty acid. Circulating cholesterol levels were twice as high in adult male +/Dsk5 mice but not in postweaned young male or female mice. The elevated total plasma cholesterol resulted mainly from an increase in low-density lipoprotein (LDL). The +/Dsk5 adult mouse liver expressed markedly reduced protein levels of LDL receptor, no change in proprotein convertase subtilisin/kexin type 9, and a markedly increased fatty acid synthase and 3-hydroxy-3-methyl-glutaryl-CoA reductase. Increased expression of transcription factors associated with enhanced cholesterol synthesis was also observed. Together, these findings suggest that the EGFR may play a regulatory role in hepatocyte proliferation and lipid metabolism in adult male mice, explaining why elevated levels of EGF or EGF-like peptides have been positively correlated to increased cholesterol levels in human studies.