Engineering living and regenerative fungal-bacterial biocomposite structures.

Engineered living materials could have the capacity to self-repair and self-replicate, sense local and distant disturbances in their environment, and respond with functionalities for reporting, actuation or remediation. However, few engineered living materials are capable of both responsivity and use in macroscopic structures. Here we describe the development, characterization and engineering of a fungal-bacterial biocomposite grown on lignocellulosic feedstocks that can form mouldable, foldable and regenerative living structures. We have developed strategies to make human-scale biocomposite structures using mould-based and origami-inspired growth and assembly paradigms. Microbiome profiling of the biocomposite over multiple generations enabled the identification of a dominant bacterial component, Pantoea agglomerans, which was further isolated and developed into a new chassis. We introduced engineered P. agglomerans into native feedstocks to yield living blocks with new biosynthetic and sensing-reporting capabilities. Bioprospecting the native microbiota to develop engineerable chassis constitutes an important strategy to facilitate the development of living biomaterials with new properties and functionalities.

Quantifying spatiotemporal variability and noise in absolute microbiota abundances using replicate sampling.

Metagenomic sequencing has enabled detailed investigation of diverse microbial communities, but understanding their spatiotemporal variability remains an important challenge. Here, we present decomposition of variance using replicate sampling (DIVERS), a method based on replicate sampling and spike-in sequencing. The method quantifies the contributions of temporal dynamics, spatial sampling variability, and technical noise to the variances and covariances of absolute bacterial abundances. We applied DIVERS to investigate a high-resolution time series of the human gut microbiome and a spatial survey of a soil bacterial community in Manhattan's Central Park. Our analysis showed that in the gut, technical noise dominated the abundance variability for nearly half of the detected taxa. DIVERS also revealed substantial spatial heterogeneity of gut microbiota, and high temporal covariances of taxa within the Bacteroidetes phylum. In the soil community, spatial variability primarily contributed to abundance fluctuations at short time scales (weeks), while temporal variability dominated at longer time scales (several months).

Scalable and cost-effective ribonuclease-based rRNA depletion for transcriptomics.

Bacterial RNA sequencing (RNA-seq) is a powerful approach for quantitatively delineating the global transcriptional profiles of microbes in order to gain deeper understanding of their physiology and function. Cost-effective bacterial RNA-seq requires efficient physical removal of ribosomal RNA (rRNA), which otherwise dominates transcriptomic reads. However, current methods to effectively deplete rRNA of diverse non-model bacterial species are lacking. Here, we describe a probe and ribonuclease based strategy for bacterial rRNA removal. We implemented the method using either chemically synthesized oligonucleotides or amplicon-based single-stranded DNA probes and validated the technique on three novel gut microbiota isolates from three distinct phyla. We further showed that different probe sets can be used on closely related species. We provide a detailed methods protocol, probe sets for >5000 common microbes from RefSeq, and an online tool to generate custom probe libraries. This approach lays the groundwork for large-scale and cost-effective bacterial transcriptomics studies.

"When to Nuss? patient age as a risk factor for complications of minimally invasive repair of pectus excavatum: a systematic review and meta-analysis".

PURPOSE: The optimal age for minimally invasive repair of pectus excavatum (MIRPE) is unclear; this study investigates the differences in complication rates among different age groups undergoing repair. METHODS: PubMed and Embase databases were searched from inception to October 2020. To assess age as a risk factor for complications, odds ratios from relevant studies were analyzed using the Mantel-Haenszel method with a random-effects model for younger vs older patients. Specific complication rates were compared between the two cohorts using a chi-squared test. RESULTS: Of the 4448 studies retrieved, 25 studies stratified complication data by age groups. From these studies, ten studies compared groups at ages < 18 and ≥ 18 and four studies compared ages < 20 and ≥ 20, and one study compared ages < 19 and ≥ 19. These fifteen studies reported on 5978 patients, with 1188 complications, for a complication rate of 19.87%. Older patients were more likely to have complications in a pooled analysis of studies comparing older vs younger patients (OR = 1.66, 95% CI = 1.28-2.14, heterogeneity I2 = 49%). Specifically, older patients were significantly more likely to experience pneumothorax, pleural effusion, wound infection, bar displacement, and reoperations. CONCLUSION: Increased age is a risk factor for complications of MIRPE. This supports repair of pectus excavatum prior to late adolescence.

Single-stage long-segment tracheal transplantation.

Tracheal transplantation has been envisioned as a viable option for reconstruction of long-segment tracheal defects. We report the first human single-stage long-segment tracheal transplantation. Narrow-band imaging and bronchoscopic biopsies demonstrate allograft vascularization and viable epithelial lining. The recipient was immunosuppressed with Tacrolimus, Mycophenolate mofetil, and corticosteroids. Six months after transplantation, the trachea is both functional and the patient is breathing without the need of a tracheostomy or stent.

Inflammation arising from obesity reduces taste bud abundance and inhibits renewal.

Despite evidence that the ability to taste is weakened by obesity and can be rescued with weight loss intervention, few studies have investigated the molecular effects of obesity on the taste system. Taste bud cells undergo continual turnover even in adulthood, exhibiting an average life span of only a few weeks, tightly controlled by a balance of proliferation and cell death. Recent data reveal that an acute inflammation event can alter this balance. We demonstrate that chronic low-grade inflammation brought on by obesity reduces the number of taste buds in gustatory tissues of mice-and is likely the cause of taste dysfunction seen in obese populations-by upsetting this balance of renewal and cell death.

Major modifications to minimize thoracic esophago-gastric leak and eradicate esophageal stricture after Ivor Lewis esophagectomy.

BACKGROUND: The Ivor Lewis esophagectomy (ILE) remains the procedure of choice for localized middle or lower esophageal carcinoma. Nevertheless, anastomotic leak remains a common complication with rates from 3% to 25% and a stricture rate as high as 40%. The frequency of these complications suggests that the procedure itself may have inherent limitations including the use of potentially ischemic tissue for the esophagogastric anastomosis. We introduce a modified technique that reduces operative steps, preserves blood supply, and uses a modified esophagogastric anastomosis. METHODS: All consecutive patients undergoing ILE with the described modified technique were identified. An esophagram was performed on postoperative day six or seven. To ensure that all cases were identified, anastomotic leaks were defined as any radiographic evidence of contrast extravasation. RESULTS: A total of 110 patients underwent the modified esophagectomy with 2 anastomotic leaks (1.82%) and zero strictures. There was 1 late death but no early deaths (<30 or 90 days) or early re-admissions (<30 days). The average number of risk factors was 2.12, and 98 patients (90%) had at least 1 risk factor in their medical history. CONCLUSIONS: The modifications proposed simplify procedural steps, limit unnecessary dissection and introduce a technique that ends the practice of connecting ischemic tissue. We believe this technique contributes to surgical durability and reduces the rate of postoperative leak and eliminates stricture.

Correction: Taste loss with obesity in mice and men.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Taste loss with obesity in mice and men.

BACKGROUND: Our sense of taste is critical in defining our food choices and habits. Located primarily in our tongue, taste buds are small assemblies of constantly renewing sensory cells, tasked with evaluating oral stimuli before the food we eat is consumed. METHODS: Using both mice and a free-living human population, we tracked taste papilla abundancy with weight gain, to test for deficiencies in the taste system of obese mice and humans with increased adiposity. RESULTS: Mice fed a high-fat diet for 8 weeks expressed markers for all subtypes of taste cells at a lower level than chow-fed counterparts. This came alongside the loss of markers for taste cell proliferation (Ki-67) and development (ß-catenin), as well as lower fungiform papillae density, consistent with earlier results showing lower circumvallate taste bud abundance in obese mice. Likewise, in a population of college students tracked through 4 years of college attendance, the change in density of fungiform papillae, which house taste buds in the anterior tongue, was negatively correlated with change in neck circumference, a marker of adiposity. CONCLUSIONS: These results highlight changes in taste during weight gain as a potentially important consideration in the study of obesity.

An Evidence-Based Approach to Management of Pectus Excavatum and Carinatum.

Pectus excavatum (PE) and pectus carinatum (PC) are the most common congenital chest wall anomalies. Current research suggests that PE and PC may result from overgrowth of the sternocostal cartilages. This can produce a deformation that displaces the sternum inward as in PE or outward as in PC. The etiology, clinical presentation, evaluation, and management of PE and PC are reviewed. Varied clinical presentations, cardiopulmonary effects, and psychosocial aspects are described.

Squamous cell carcinoma-related oncogene (SCCRO) neddylates Cul3 protein to selectively promote midbody localization and activity of Cul3KLHL21 protein complex during abscission.

Squamous cell carcinoma-related oncogene (SCCRO)/DCUN1D1, a component of the neddylation E3 complex, regulates the activity of the cullin-RING-ligase type of ubiquitination E3s by promoting neddylation of cullin family members. Studies have shown that SCCRO regulates proliferation in vitro and in vivo Here we show that inactivation of SCCRO results in prolonged mitotic time because of delayed and/or failed abscission. The effects of SCCRO on abscission involve its role in neddylation and localization of Cul3 to the midbody. The Cul3 adaptor KLHL21 mediates the effects of SCCRO on abscission, as it fails to localize to the midbody in SCCRO-deficient cells during abscission, and its inactivation resulted in phenotypic changes identical to SCCRO inactivation. Ubiquitination-promoted turnover of Aurora B at the midbody was deficient in SCCRO- and KLHL21-deficient cells, suggesting that it is the target of Cul3KLHL21 at the midbody. Correction of abscission delays in SCCRO-deficient cells with addition of an Aurora B inhibitor at the midbody stage suggests that Aurora B is the target of SCCRO-promoted Cul3KLHL21 activity. The activity of other Cul3-anchored complexes, including Cul3KLHL9/KLHL13, was intact in SCCRO-deficient cells, suggesting that SCCRO selectively, rather than collectively, neddylates cullins in vivo Combined, these findings support a model in which the SCCRO, substrate, and substrate adaptors cooperatively provide tight control of neddylation and cullin-RING-ligase activity in vivo.

Correction to: Baseline and annual repeat rounds of screening: implications for optimal regimens of screening.

The original version of this article unfortunately contained a mistake. The conflict of interest was incorrect.

Baseline and annual repeat rounds of screening: implications for optimal regimens of screening.

OBJECTIVES: Differences in results of baseline and subsequent annual repeat rounds provide important information for optimising the regimen of screening. METHODS: A prospective cohort study of 65,374 was reviewed to examine the frequency/percentages of the largest noncalcified nodule (NCN), lung cancer cell types and Kaplan-Meier (K-M) survival rates, separately for baseline and annual rounds. RESULTS: Of 65,374 baseline screenings, NCNs were identified in 28,279 (43.3%); lung cancer in 737 (1.1%). Of 74,482 annual repeat screenings, new NCNs were identified in 4959 (7%); lung cancer in 179 (0.24%). Only adenocarcinoma was diagnosed in subsolid NCNs. Percentages of lung cancers by cell type were significantly different (p < 0.0001) in the baseline round compared with annual rounds, reflecting length bias, as were the ratios, reflecting lead times. Long-term K-M survival rate was 100% for typical carcinoids and for adenocarcinomas manifesting as subsolid NCNs; 85% (95% CI 81-89%) for adenocarcinoma, 74% (95% CI 63-85%) for squamous cell, 48% (95% CI 34-62%) for small cell. The rank ordering by lead time was the same as the rank ordering by survival rates. CONCLUSIONS: The significant differences in the frequency of NCNs and frequency and aggressiveness of diagnosed cancers in baseline and annual repeat need to be recognised for an optimal regimen of screening. KEY POINTS: • Lung cancer aggressiveness varies considerably by cell type and nodule consistency. • Kaplan-Meier survival rates varied by cell type between 100% and 48%. • The percentages of lung cancers by cell type in screening rounds reflect screening biases. • Rank ordering by cell type survival is consistent with that by lead times. • Empirical evidence provides critical information for the regimen of screening.

IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype.

Both genome-wide genetic and epigenetic alterations are fundamentally important for the development of cancers, but the interdependence of these aberrations is poorly understood. Glioblastomas and other cancers with the CpG island methylator phenotype (CIMP) constitute a subset of tumours with extensive epigenomic aberrations and a distinct biology. Glioma CIMP (G-CIMP) is a powerful determinant of tumour pathogenicity, but the molecular basis of G-CIMP remains unresolved. Here we show that mutation of a single gene, isocitrate dehydrogenase 1 (IDH1), establishes G-CIMP by remodelling the methylome. This remodelling results in reorganization of the methylome and transcriptome. Examination of the epigenome of a large set of intermediate-grade gliomas demonstrates a distinct G-CIMP phenotype that is highly dependent on the presence of IDH mutation. Introduction of mutant IDH1 into primary human astrocytes alters specific histone marks, induces extensive DNA hypermethylation, and reshapes the methylome in a fashion that mirrors the changes observed in G-CIMP-positive lower-grade gliomas. Furthermore, the epigenomic alterations resulting from mutant IDH1 activate key gene expression programs, characterize G-CIMP-positive proneural glioblastomas but not other glioblastomas, and are predictive of improved survival. Our findings demonstrate that IDH mutation is the molecular basis of CIMP in gliomas, provide a framework for understanding oncogenesis in these gliomas, and highlight the interplay between genomic and epigenomic changes in human cancers.

Squamous Cell Carcinoma-related Oncogene (SCCRO) Family Members Regulate Cell Growth and Proliferation through Their Cooperative and Antagonistic Effects on Cullin Neddylation.

SCCRO (squamous cell carcinoma-related oncogene; also known as DCUN1D1) is a highly conserved gene that functions as an E3 in neddylation. Although inactivation of SCCRO in yeast results in lethality, SCCRO(-/-) mice are viable. The exclusive presence of highly conserved paralogues in higher organisms led us to assess whether compensation by SCCRO paralogues rescues lethality in SCCRO(-/-) mice. Using murine and Drosophila models, we assessed the in vivo activities of SCCRO and its paralogues in cullin neddylation. We found that SCCRO family members have overlapping and antagonistic activity that regulates neddylation and cell proliferation activities in vivo. In flies, both dSCCRO and dSCCRO3 promote neddylation and cell proliferation, whereas dSCCRO4 negatively regulates these processes. Analysis of somatic clones showed that the effects that these paralogues have on proliferation serve to promote cell competition, leading to apoptosis in clones with a net decrease in neddylation activity. We found that dSCCRO and, to a lesser extent, dSCCRO3 rescue the neddylation and proliferation defects promoted by expression of SCCRO4. dSCCRO and dSCCRO3 functioned cooperatively, with their coexpression resulting in an increase in both the neddylated cullin fraction and proliferation activity. In contrast, human SCCRO and SCCRO4 promote, and human SCCRO3 inhibits, neddylation and proliferation when expressed in flies. Our findings provide the first insights into the mechanisms through which SCCRO family members cooperatively regulate neddylation and cell proliferation.

Dorsal root ganglion transcriptome analysis following peripheral nerve injury in mice.

BACKGROUND: Peripheral nerve injury leads to changes in gene expression in primary sensory neurons of the injured dorsal root ganglia. These changes are believed to be involved in neuropathic pain genesis. Previously, these changes have been identified using gene microarrays or next generation RNA sequencing with poly-A tail selection, but these approaches cannot provide a more thorough analysis of gene expression alterations after nerve injury. METHODS: The present study chose to eliminate mRNA poly-A tail selection and perform strand-specific next generation RNA sequencing to analyze whole transcriptomes in the injured dorsal root ganglia following spinal nerve ligation. Quantitative real-time reverse transcriptase polymerase chain reaction assay was carried out to verify the changes of some differentially expressed RNAs in the injured dorsal root ganglia after spinal nerve ligation. RESULTS: Our results showed that more than 50 million (M) paired mapped sequences with strand information were yielded in each group (51.87 M-56.12 M in sham vs. 51.08 M-57.99 M in spinal nerve ligation). Six days after spinal nerve ligation, expression levels of 11,163 out of a total of 27,463 identified genes in the injured dorsal root ganglia significantly changed, of which 52.14% were upregulated and 47.86% downregulated. The largest transcriptional changes were observed in protein-coding genes (91.5%) followed by noncoding RNAs. Within 944 differentially expressed noncoding RNAs, the most significant changes were seen in long interspersed noncoding RNAs followed by antisense RNAs, processed transcripts, and pseudogenes. We observed a notable proportion of reads aligning to intronic regions in both groups (44.0% in sham vs. 49.6% in spinal nerve ligation). Using quantitative real-time polymerase chain reaction, we confirmed consistent differential expression of selected genes including Kcna2, Oprm1 as well as lncRNAs Gm21781 and 4732491K20Rik following spinal nerve ligation. CONCLUSION: Our findings suggest that next generation RNA sequencing can be used as a promising approach to analyze the changes of whole transcriptomes in dorsal root ganglia following nerve injury and to possibly identify new targets for prevention and treatment of neuropathic pain.

Racial disparities in esophageal cancer survival after surgery.

OBJECTIVES: Esophageal cancer (EC) black patients have higher mortality rates than Whites. The lower rate of surgery in Blacks may explain the survival difference. We explored the Surveillance Epidemiology and End Results database to determine the impact of surgery on mortality in Blacks and Whites EC. METHODS: All cases of pathologically proven local and locoregional adenocarcinoma and squamous cell carcinoma of the esophagus from 1973 to 2011 were identified (13,678 White, 2,894 Black patients). Cervical esophageal cancer was excluded. Age, sex, diagnosis year, stage, cancer-directed surgery, radiation, and vital status were analyzed according to self-reported race. RESULTS: Blacks had higher 1-year mortality, adjusted for age, sex, stage, year of diagnosis, histology, and therapy [adjusted hazard ratio (HRadj ): 1.24 (95% CI 1.16-1.32)]. Undergoing surgery was an independent predictor of improved survival overall (HRadj 0.30, 95% CI 0.27-0.33). Black patients treated surgically experienced significantly lower survival than Whites, but the difference was not observed in those who did not undergo surgery. CONCLUSIONS: Although surgery appears to reduce mortality overall, early survival is worse for Blacks. Investigation into racial disparities in health care access and delivery, and to skilled esophageal surgeons is warranted to improve survival for all patients, particularly Blacks. J. Surg. Oncol. 2016;113:659-664. © 2016 Wiley Periodicals, Inc.

Housing Quality in a Randomized Controlled Trial of Housing First for Homeless Individuals with Mental Illness: Correlates and Associations with Outcomes.

Housing quality (HQ) is associated with mental health, and may mediate outcomes in housing interventions. However, studies of housing interventions rarely report HQ. The purpose of this study was to describe HQ in a multi-site randomized controlled trial of Housing First (HF) in five Canadian cities and to examine possible differences by treatment group (HF recipients and treatment-as-usual (TAU) participants who were able to find housing through other programs or on their own). We also examined the association between HQ and the primary trial outcome: housing stability. The performance of a new multi-dimensional standardized observer-rated housing quality scale (the OHQS) in a relatively large cross-site sample was also of interest. HQ was rated by trained research assistants for 204 HF participants and 228 TAU participants using the OHQS. General linear regression models were used to examine unit/building quality scores by group and site adjusting for other group differences, and as a predictor of housing stability outcomes after 24 months of follow-up. The OHQS was found to have good reliability and validity, but because most of the neighborhood subscale items were negatively correlated with the overall scale, only unit and building items were included in the total HQ score (possible scores ranging from 13.5 to 135). Unit/building HQ was significantly better for the HF group overall (91.2 (95 % CI = 89.6-92.9) vs. 88.3 (95 % CI = 86.1-90.5); p = .036), and in one site. HQ in the TAU group was much more variable than the HF group overall (W (mean) = 24.7; p < .001) and in four of five sites. Unit/building HQ scores were positively associated with housing stability: (73.4 (95 % CI 68.3-78.5) for those housed none of the time; 91.1 (95 % CI 89.2-93.0) for those housed some of the time; and 93.1 (95 % CI 91.4-94.9)) for those housed all of the time (F = 43.9 p < .001). This association held after adjusting for site, housing characteristics, participant ethnocultural status, community functioning, and social support. This study demonstrates that HQ can be as good or better, and less variable, in HF programs in Canada that systematically and predominantly source housing stock from the private sector compared to housing procured outside of an HF program. HQ is also an important predictor of housing stability outcomes.

Short-term pre- and post-operative stress prolongs incision-induced pain hypersensitivity without changing basal pain perception.

BACKGROUND: Chronic stress has been reported to increase basal pain sensitivity and/or exacerbate existing persistent pain. However, most surgical patients have normal physiological and psychological health status such as normal pain perception before surgery although they do experience short-term stress during pre- and post-operative periods. Whether or not this short-term stress affects persistent postsurgical pain is unclear. RESULTS: In this study, we showed that pre- or post-surgical exposure to immobilization 6 h daily for three consecutive days did not change basal responses to mechanical, thermal, or cold stimuli or peak levels of incision-induced hypersensitivity to these stimuli; however, immobilization did prolong the duration of incision-induced hypersensitivity in both male and female rats. These phenomena were also observed in post-surgical exposure to forced swimming 25 min daily for 3 consecutive days. Short-term stress induced by immobilization was demonstrated by an elevation in the level of serum corticosterone, an increase in swim immobility, and a decrease in sucrose consumption. Blocking this short-term stress via intrathecal administration of a selective glucocorticoid receptor antagonist, RU38486, or bilateral adrenalectomy significantly attenuated the prolongation of incision-induced hypersensitivity to mechanical, thermal, and cold stimuli. CONCLUSION: Our results indicate that short-term stress during the pre- or post-operative period delays postoperative pain recovery although it does not affect basal pain perception. Prevention of short-term stress may facilitate patients' recovery from postoperative pain.