Metabolic liver cancer: associations of rare and common germline variants in one-carbon metabolism and DNA methylation genes.

Animal studies implicate one-carbon metabolism and DNA methylation genes in hepatocellular carcinoma (HCC) development in the setting of metabolic perturbations. Using human samples, we investigated the associations between common and rare variants in these closely related biochemical pathways and risk for metabolic HCC development in a multicenter international study. We performed targeted exome sequencing of 64 genes among 556 metabolic HCC cases and 643 cancer-free controls with metabolic conditions. Multivariable logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for multiple comparisons. Gene-burden tests were used for rare variant associations. Analyses were performed in the overall sample and among non-Hispanic whites. The results show that among non-Hispanic whites, presence of rare functional variants in ABCC2 was associated with 7-fold higher risk of metabolic HCC (OR = 6.92, 95% CI: 2.38-20.15, P = 0.0004), and this association remained significant when analyses were restricted to functional rare variants observed in ≥2 participants (cases 3.2% versus controls 0.0%, P = 1.02 × 10-5). In the overall multiethnic sample, presence of rare functional variants in ABCC2 was nominally associated with metabolic HCC (OR = 3.60, 95% CI: 1.52-8.58, P = 0.004), with similar nominal association when analyses were restricted to functional rare variants observed in ≥2 participants (cases 2.9% versus controls 0.2%, P = 0.006). A common variant in PNPLA3 (rs738409[G]) was associated with higher HCC risk in the overall sample (P = 6.36 × 10-6) and in non-Hispanic whites (P = 0.0002). Our findings indicate that rare functional variants in ABCC2 are associated with susceptibility to metabolic HCC in non-Hispanic whites. PNPLA3-rs738409 is also associated with metabolic HCC risk.

HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia.

BACKGROUND & AIMS: The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. METHODS: The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpdafl/fl) and hepatocyte-specific Hilpda knockout (HilpdaΔHep) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. RESULTS: In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, HilpdaΔHep exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. CONCLUSIONS: Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. IMPACT AND IMPLICATIONS: Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.

A Novel Approach to Limb Salvage: Healing Transmetatarsal Amputations without a Viable Plantar Flap.

BACKGROUND: The lack of a viable plantar flap in patients undergoing transmetatarsal amputation has been considered an indication for below-knee amputation (BKA). In an effort to reduce limb loss in this patient population, we sought to review our experience with transmetatarsal amputation salvage in patients with an open, guillotine transmetatarsal amputation. We hypothesized that performing a transmetatarsal amputation without a viable flap would extend time of independent ambulation and improve limb salvage. METHODS: This is a retrospective review of 27 consecutive patients who did not have a viable plantar flap and who underwent an open, guillotine transmetatarsal amputation. Patients presented with a nonviable plantar flap due to either extensive tissue loss on initial presentation, or secondary transmetatarsal amputation (TMA) flap necrosis. Patients initially underwent an open, guillotine TMA for control of infection and debridement of nonviable tissue. To achieve best results, during procedure, the metatarsals were resected to be as flush with soft tissue as possible. Once infection was resolved and all nonviable tissue debrided, negative pressure wound therapy (NPWT) was applied to the open wound. NPWT was continued until a base of granulation tissue covered the previously exposed bone. Wound closure was obtained by either the application of a split-thickness skin graft (STSG) or through continued NPWT allowing the wound to heal by secondary intention. RESULTS: Between January 2016 and December 2018, there were 27 open TMAs performed in 27 patients. Two patients did not granulate sufficiently and underwent BKA. Fourteen patients underwent STSG for closure, whereas 11 patients continued with NPWT. In the STSG group, 12 (86%) of the patients are healed, with a median time to complete healing of 75 days (range 28-330 days); the remaining 2 are ambulatory and undergoing continued wound care. In the 11 patients who did not receive STSG, 7 (64%) are healed with a median time to heal of 165 days. Of the remaining 4 patients in this group, 3 are ambulatory and still undergoing wound care, one was lost to follow-up. Overall, 19 patients (70%) have completely healed with a median time to heal of 82 days. CONCLUSIONS: Limb salvage in patients with a nonviable plantar flap for TMA is possible and should be a considered procedure. This technique has the potential to improve functional outcomes and limb salvage in patients who might otherwise undergo BKA.

Multidisciplinary Limb Salvage Service Reduces Major Amputations in Diabetic Foot Infections.

BACKGROUND: Diabetic foot infections (DFIs) can lead to limb loss and mortality. To improve patient care at a safety-net teaching hospital, we created a multidisciplinary limb salvage service (LSS). METHODS: We recruited a cohort prospectively and compared it to a historical control group. Adults admitted to the newly established LSS for DFI during a 6-month period from 2016 to 2017 were included prospectively. Patients admitted to the LSS had routine endocrine and infectious diseases consultations according to a standardized protocol. A retrospective analysis of patients admitted to the acute care surgical service for DFI before creation of the LSS during an 8-month period from 2014 to 2015 was performed. RESULTS: A total of 250 patients were divided into two groups: the pre-LSS (n = 92) and the LSS (n = 158) groups. There were no significant differences in baseline characteristics. Although all patients were ultimately diagnosed with diabetes, more patients in the LSS group had hypertension (71% versus 56%; P = .01) and a prior diagnosis of diabetes mellitus (92% versus 63%; P < .001) compared to the pre-LSS group. Significantly, with the LSS, fewer patients underwent a below-the-knee amputation (3.6% versus 13%; P = .001). There was no difference in the length of hospital stay or 30-day readmission rate between the groups. Further broken down into Hispanic versus non-Hispanic, we noted that Hispanics had significantly lower rates of below-the-knee amputations (3.6% versus 13.0%; P = .02) in the LSS cohort. CONCLUSIONS: The initiation of a multidisciplinary LSS decreased the below-the-knee amputation rate in patients with DFIs. Length of stay was not increased, nor was the 30-day readmission rate affected. These results suggest that a robust multidisciplinary LSS dedicated to the management of DFIs is both feasible and effective, even in safety-net hospitals.

Genomic epidemiology reveals the dominance of Hennepin County in transmission of SARS-CoV-2 in Minnesota from 2020-2022.

SARS-CoV-2 has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of virus evolution and spread, and to inform policy decisions. We sequenced viral genomes from over 22,000 patient samples tested at Mayo Clinic Laboratories between 2020-2022 and use Bayesian phylodynamics to describe county and regional spread in Minnesota. The earliest introduction into Minnesota was to Hennepin County from a domestic source around January 22, 2020; six weeks before the first confirmed case in the state. This led to the virus spreading to Northern Minnesota, and eventually, the rest of the state. International introductions were most abundant in Hennepin (home to the Minneapolis/St. Paul International (MSP) airport) totaling 45 (out of 107) over the two-year period. Southern Minnesota counties were most common for domestic introductions with 19 (out of 64), potentially driven by bordering states such as Iowa and Wisconsin as well as Illinois which is nearby. Hennepin also was, by far, the most dominant source of in-state transmissions to other Minnesota locations (n=772) over the two-year period. We also analyzed the diversity of the location source of SARS-CoV-2 viruses in each county and noted the timing of state-wide policies as well as trends in clinical cases. Neither the number of clinical cases or major policy decisions, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, appeared to have impact on virus diversity across each individual county.

Genomic epidemiology reveals the dominance of Hennepin County in the transmission of SARS-CoV-2 in Minnesota from 2020 to 2022.

IMPORTANCE: We analyzed over 22,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes of patient samples tested at Mayo Clinic Laboratories during a 2-year period in the COVID-19 pandemic, which included Alpha, Delta, and Omicron variants of concern to examine the roles and relationships of Minnesota virus transmission. We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after an initial predicted introduction with the virus in early 2020 from an international source, while other counties acted as transmission "sinks." In addition, major policies, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties.

Automation of hybridization and capture based next generation sequencing library preparation requires reduction of on-deck bead binding and heated wash temperatures.

Capture-based library preparation for next generation sequencing (NGS) offers a balance between sequencing depth and bioinformatics cost of analysis. Liquid handling automation enhances the reliability of the library preparation process by reducing sample-to-sample variation and substantially enhances throughput, particularly when it can be employed in a 'walk-away' fashion with limited hands-on interaction. This requires complex series of mixing and heating steps like those utilized in capture chemistries to happen on the liquid handler. While developing liquid handling automation for Integrated DNA Technologies (IDT) xGen Exome, Illumina TruSight Oncology 500, and Personal Genome Diagnostics (PGDx) elio Plasma Resolve chemistries on the PerkinElmer Sciclone liquid handler, we found that applying the capture temperatures recommended for manual library preparation results in low yield on automation. To restore the final library yield, we reduced bead binding and/or heated wash temperatures of the Peltier heaters on the liquid handlers by about 10°C. Since this applied across three unique capture-based chemistries, we consider this a generalizable principle of automating capture on the Sciclone. We hypothesize that this is driven by the very different thermodynamic environments represented by a sealed plate on a thermal cycler and a plate with a lid on a Peltier heater. This phenomenon should be considered when automating NGS library preparation on PerkinElmer Sciclone instruments.

Use of surface-enhanced laser desorption/ionization -time of flight to explore bacterial proteomes.

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is a recent technology that allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. We compared three simple procedures for protein extraction from bacteria for compatibility with the ProteinChip Array; we also determined the amount of protein required for each analysis. The protocol for the SELDI analysis was evaluated by generating protein expression profiles of a Streptococcus pneumoniae strain grown in different conditions and those of different strains of the same species. The protocol also was successfully applied to a wide range of Gram positive and negative bacteria. The results of this study suggest the appropriateness of this technology for microorganism protein profiling as complementary or alternative to two-dimensional gel electrophoresis.