Mapping Spatial Genetic Landscapes in Tissue Sections through Microscale Integration of Sampling Methodology into Genomic Workflows.

In cancer research, genomic profiles are often extracted from homogenized macrodissections of tissues, with the histological context lost and a large fraction of material underutilized. Pertinently, the spatial genomic landscape provides critical complementary information in deciphering disease heterogeneity and progression. Microscale sampling methods such as microdissection to obtain such information are often destructive to a sizeable fraction of the biopsy sample, thus showing limited multiplexability and adaptability to different assays. A modular microfluidic technology is here implemented to recover cells at the microscale from tumor tissue sections, with minimal disruption of unsampled areas and tailored to interface with genome profiling workflows, which is directed here toward evaluating intratumoral genomic heterogeneity. The integrated workflow-GeneScape-is used to evaluate heterogeneity in a metastatic mammary carcinoma, showing distinct single nucleotide variants and copy number variations in different tumor tissue regions, suggesting the polyclonal origin of the metastasis as well as development driven by multiple location-specific drivers.

Vein graft aneurysms following popliteal aneurysm repair are more common than we think.

True infrainguinal vein graft aneurysms are reported infrequently in the literature. We sought to identify the true incidence of these graft aneurysms after popliteal aneurysm repair and identify factors which may increase the risk of such aneurysms developing. Using a prospectively compiled database, we identified patients who underwent a popliteal aneurysm repair between January 1996 and January 2011 at a single district general hospital. Patients were routinely followed up in a graft surveillance programme. Out of 45 patients requiring repair of a popliteal aneurysm over a 15-year period, four (8.8%) patients developed aneurysmal graft disease. Of the patients who developed graft aneurysms, all had aneurysmal disease at other sites compared with 18 (45.0%) patients who did not develop graft aneurysms. Patients with graft aneurysms had a mean of 1.60 aneurysms elsewhere compared to 0.58 in patients with non-aneurysmal grafts (P = 0.005). True vein graft aneurysms occur in a significant number of patients following popliteal aneurysm repair. Our data would suggest this to be more likely in patients who have aneurysms elsewhere and therefore a predisposition to aneurysmal disease. It may be appropriate for patients with aneurysms at other sites to undergo more prolonged post-operative graft surveillance.

Multilevel bypass grafting: is it worth it?

BACKGROUND: Traditionally, multilevel arterial disease has been treated with an inflow procedure only but simultaneous multilevel bypass graft procedures have been attempted. However, these procedures are potentially high risk. We report our single-center experience of performing multilevel bypass grafts over the last 15 years. METHODS: We retrospectively identified patients undergoing simultaneous aortoiliac and infrainguinal bypasses between January 1996 and January 2011 at a single district general hospital. RESULTS: There were 32 multilevel procedures performed. Indication for surgery was acute ischemia in 10 (31.3%), critical ischemia without tissue loss in 10 (31.3%), with tissue loss in 10 (31.3%), and claudication in 2 (6.3%). In 23 (71.9%) cases inflow was restored using a direct iliofemoral or aortofemoral reconstruction. In the remaining 9 (28.1%), an extra-anatomic bypass was constructed. Two (6.3%) patients died within 30 days of surgery. Twenty-nine (90.6%) patients survived to discharge. Twenty-eight patients (87.5%) were alive 1 year after surgery. Limb salvage was 96.9%, 85.7%, and 75.9% at 30 days, 1 year, and 5 years, respectively. Twelve (37.5%) patients required a total of 19 further ipsilateral vascular procedures. CONCLUSIONS: Our results demonstrate that multilevel bypass procedures can be performed with good long-term outcomes and acceptable mortality, in what is typically a high-risk group with extensive comorbidities. In patients with severe critical limb ischaemia and tissue loss, who have a combination of aortoiliac and infrainguinal disease, there are significant benefits to a primary multilevel grafting procedure.

Quantification of the spread of SARS-CoV-2 variant B.1.1.7 in Switzerland.

BACKGROUND: In December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40-80 % (Volz et al., 2021; Leung et al., 2021; Davies et al., 2021). AIM: This study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland. METHODS: We generated whole genome sequences from 11.8 % of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant's transmission fitness advantage on a national and a regional scale. RESULTS: We estimate B.1.1.7 had a transmission fitness advantage of 43-52 % compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07-1.41] from 01 January until 17 January 2021 and 1.18 [1.06-1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00-1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021. CONCLUSION: The observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2-3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.

Patency of infra-inguinal vein grafts--effect of intraoperative Doppler assessment and a graft surveillance program.

OBJECTIVE: To assess the value of intraoperative graft flow and resistance measurements and a graft surveillance program to predict at-risk infra-inguinal bypass grafts. METHODS: Four hundred sixty-eight infra-inguinal bypass procedures performed between 1995-2006 underwent intraoperative measurement of graft flow and resistance using a Scimed OpDop. These data were correlated with graft outcome at six weeks. Two hundred fifty-four (73%) grafts were entered into a graft surveillance program and the effect of this on primary-assisted graft patency was assessed. RESULTS: Overall primary and primary-assisted graft patency was 81% and 83% at six weeks and 42% and 64% at three years. Grafts failing by six weeks had significantly lower flow (130.5 mL/min vs. 150.5 mL/min, P = .009) and higher resistance (0.67 peripheral resistance units (PRU) vs. 0.57 PRU, P = .004) than those remaining patent. However, OpDop measured flow and resistance was a poor predictor of graft failure in individual cases (area under ROC curve, 0.57). While there was no statistical difference in primary 18-month patency rates between grafts undergoing surveillance and those undergoing clinical follow up (55% vs. 76%, P = .133), primary-assisted 18-month patency rates were significantly higher in the surveillance group (83% vs. 77%, P = .042). CONCLUSION: Intraoperative measurements of graft flow and resistance do not predict graft outcome at six weeks. However, surveillance does identify at-risk grafts and improves mid-term primary-assisted patency.

Iliofemoral pulsion endarterectomy.

We present our experience with a technique of endarterectomy for use in patients with iliofemoral occlusive disease, in which the atheromatous plug is extruded from the intact artery by external manipulation (pulsion). A retrospective review of consecutive patients who underwent surgical iliofemoral pulsion endarterectomy (IFPE) in two vascular surgery units between 1998 and 2006 was performed. Primary and secondary graft patency, limb salvage, and patient survival rates were determined using Kaplan-Meier methods. Fifty-eight IFPEs were carried out successfully on 54 patients (36 men, 18 women, median age 66 years) presenting with critical limb ischemia (n=23), with claudication (n=29), or in conjunction with abdominal aortic aneurysm repair (n=6). Mean (range) follow-up was 17 months (1-69). During this period six patients (all male, mean age 64 years) underwent iliofemoral bypass using a prosthetic graft when the iliac arteries were found unsuitable for endarterectomy because of hypoplasia or heavy calcification. Two-year cumulative primary patency of IFPE was 95%, secondary patency 100%, limb salvage 98.5%, and patient survival 73%. This modification of iliac endarterectomy is a relatively simple and safe technique that eschews prosthetics and offers a durable solution for the majority of patients with extensive iliofemoral occlusive disease.

Genetic Loci Governing Androgenic Capacity in Perennial Ryegrass (Lolium perenne L.).

Immature pollen can be induced to switch developmental pathways from gametogenesis to embryogenesis and subsequently regenerate into homozygous, diploid plants. Such androgenic production of doubled haploids is particularly useful for species where inbreeding is hampered by effective self-incompatibility systems. Therefore, increasing the generally low androgenic capacity of perennial ryegrass (Lolium perenne L.) germplasm would enable the efficient production of homozygous plant material, so that a more effective exploitation of heterosis through hybrid breeding schemes can be realized. Here, we present the results of a genome-wide association study in a heterozygous, multiparental population of perennial ryegrass (n = 391) segregating for androgenic capacity. Genotyping-by-sequencing was used to interrogate gene- dense genomic regions and revealed over 1,100 polymorphic sites. Between one and 10 quantitative trait loci (QTL) were identified for anther response, embryo and total plant production, green and albino plant production and regeneration. Most traits were under polygenic control, although a major QTL on linkage group 5 was associated with green plant regeneration. Distinct genetic factors seem to affect green and albino plant recovery. Two intriguing candidate genes, encoding chromatin binding domains of the developmental phase transition regulator, Polycomb Repressive Complex 2, were identified. Our results shed the first light on the molecular mechanisms behind perennial ryegrass microspore embryogenesis and enable marker-assisted introgression of androgenic capacity into recalcitrant germplasm of this forage crop of global significance.

In Silico Identification of Candidate Genes for Fertility Restoration in Cytoplasmic Male Sterile Perennial Ryegrass (Lolium perenne L.).

Perennial ryegrass (Lolium perenne L.) is widely used for forage production in both permanent and temporary grassland systems. To increase yields in perennial ryegrass, recent breeding efforts have been focused on strategies to more efficiently exploit heterosis by hybrid breeding. Cytoplasmic male sterility (CMS) is a widely applied mechanism to control pollination for commercial hybrid seed production and although CMS systems have been identified in perennial ryegrass, they are yet to be fully characterized. Here, we present a bioinformatics pipeline for efficient identification of candidate restorer of fertility (Rf) genes for CMS. From a high-quality draft of the perennial ryegrass genome, 373 pentatricopeptide repeat (PPR) genes were identified and classified, further identifying 25 restorer of fertility-like PPR (RFL) genes through a combination of DNA sequence clustering and comparison to known Rf genes. This extensive gene family was targeted as the majority of Rf genes in higher plants are RFL genes. These RFL genes were further investigated by phylogenetic analyses, identifying three groups of perennial ryegrass RFLs. These three groups likely represent genomic regions of active RFL generation and identify the probable location of perennial ryegrass PPR-Rf genes. This pipeline allows for the identification of candidate PPR-Rf genes from genomic sequence data and can be used in any plant species. Functional markers for PPR-Rf genes will facilitate map-based cloning of Rf genes and enable the use of CMS as an efficient tool to control pollination for hybrid crop production.