Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Geranylgeranyl generosity: a new prenyl-transferase gives a fat to a SNARE protein.

Protein prenylation, a well-defined protein consensus motifs direct modification by one of three prenyl-transferases, has been an area of fairly settled science for 20 or 30 years. Protein prenylation, the specific prenyl modification (farnesyl or geranylgeranyl), as well as the prenyl-transferases involved can be inferred by protein sequence. Two new papers now upset this settled wisdom with the discovery of a fourth prenyl-transferase, namely geranylgeranyl-transferase-III (GGTase-III) (Kuchay et al, 2019; Shirakawa et al, 2020).

Elite rhetoric can undermine democratic norms.

Democratic stability depends on citizens on the losing side accepting election outcomes. Can rhetoric by political leaders undermine this norm? Using a panel survey experiment, we evaluate the effects of exposure to multiple statements from former president Donald Trump attacking the legitimacy of the 2020 US presidential election. Although exposure to these statements does not measurably affect general support for political violence or belief in democracy, it erodes trust and confidence in elections and increases belief that the election is rigged among people who approve of Trump's job performance. These results suggest that rhetoric from political elites can undermine respect for critical democratic norms among their supporters.

Ultrasound Evaluation of Gastric Emptying Time in Healthy Term Neonates after Formula Feeding.

BACKGROUND: The current American Society of Anesthesiologists fasting guideline for formula-fed infants in the periprocedural setting is 6 h. Prolonged fasting in very young infants is associated with an increased risk for hypoglycemia and dehydration as well as patient discomfort and patient/parental dissatisfaction. This study aimed to determine the time to gastric emptying in healthy neonates after formula feeding by serially evaluating the gastric antrum with ultrasound. The authors hypothesized that gastric emptying times in formula-fed neonates are significantly shorter than the current 6 h fasting recommendation. METHODS: After institutional review board approval and written informed parental consent, ultrasound examination was performed in healthy full-term neonates before and after formula feeding at 15-min intervals until return to baseline. Ultrasound images of the gastric antrum were measured to obtain cross-sectional areas, which were then used to estimate gastric antral volumes. RESULTS: Forty-six of 48 recruited neonates were included in the final analysis. Gastric emptying times ranged from 45 to 150 min and averaged 92.9 min (95% CI, 80.2 to 105.7 min; 99% CI, 76.0 to 109.8 min) in the overall study group. No significant differences were found in times to gastric emptying between male and female neonates (male: mean, 93.3 [95% CI, 82.4 to 104.2 min]; female: mean, 92.6 [95% CI, 82.0 to 103.2 min]; P = 0.930) or those delivered by vaginal versus cesarean routes (vaginal: mean, 93.9 [95% CI, 81.7 to 106.1 min]; cesarean: mean, 92.2 [95% CI, 82.5 to 101.9 min]; P = 0.819). CONCLUSIONS: These results demonstrate that gastric emptying times are substantially less than the current fasting guideline of 6 h for formula-fed, healthy term neonates.

Executive functions in children with heart disease: a systematic review and meta-analysis.

CONTEXT: People with CHD are at increased risk for executive functioning deficits. Meta-analyses of these measures in CHD patients compared to healthy controls have not been reported. OBJECTIVE: To examine differences in executive functions in individuals with CHD compared to healthy controls. DATA SOURCES: We performed a systematic review of publications from 1 January, 1986 to 15 June, 2020 indexed in PubMed, CINAHL, EMBASE, PsycInfo, Web of Science, and the Cochrane Library. STUDY SELECTION: Inclusion criteria were (1) studies containing at least one executive function measure; (2) participants were over the age of three. DATA EXTRACTION: Data extraction and quality assessment were performed independently by two authors. We used a shifting unit-of-analysis approach and pooled data using a random effects model. RESULTS: The search yielded 61,217 results. Twenty-eight studies met criteria. A total of 7789 people with CHD were compared with 8187 healthy controls. We found the following standardised mean differences: -0.628 (-0.726, -0.531) for cognitive flexibility and set shifting, -0.469 (-0.606, -0.333) for inhibition, -0.369 (-0.466, -0.273) for working memory, -0.334 (-0.546, -0.121) for planning/problem solving, -0.361 (-0.576, -0.147) for summary measures, and -0.444 (-0.614, -0.274) for reporter-based measures (p < 0.001). LIMITATIONS: Our analysis consisted of cross-sectional and observational studies. We could not quantify the effect of collinearity. CONCLUSIONS: Individuals with CHD appear to have at least moderate deficits in executive functions. Given the growing population of people with CHD, more attention should be devoted to identifying executive dysfunction in this vulnerable group.

Redefining the Trauma Triage Matrix: The Role of Emergent Interventions.

BACKGROUND: A tiered trauma team activation (TTA) system aims to allocate resources proportional to the patient's need based upon injury burden. The current metrics used to evaluate appropriateness of TTA are the trauma triage matrix (TTM), need for trauma intervention (NFTI), and secondary triage assessment tool (STAT). MATERIALS AND METHODS: In this retrospective study, we compared the effectiveness of the need for an emergent intervention within 6 h (NEI-6) with existing definitions. Data from the Michigan Trauma Quality Improvement Program was utilized. The dataset contains information from 31 level 1 and 2 trauma centers from 2011 to 2017. Inclusion criteria were: adult patients (≥16 y) and ISS ≥5. RESULTS: 73,818 patients were included in the study. Thirty percentage of trauma patients met criteria for STAT, 21% for NFTI, 20% for TTM, and 13% for NEI-6. NEI-6 was associated with the lowest rate of undertriage at 6.5% (STAT 22.3%, NFTI 14.0%, TTM 14.3%). NEI-6 best predicted undertriage mortality, early mortality, in-hospital mortality, and late (>60 h) mortality. Most patients who met criteria for TTM (58%), NFTI (51%), and STAT (62%) did not require emergent intervention. All four methods had similar rates of early mortality for patients who did not meet criteria (0.3%-0.5%). CONCLUSIONS: NEI-6 performs better than TTM, NFTI, and STAT in terms of undertriage, mortality and need for resource utilization. Other methods resulted in significantly more full TTAs than NEI-6 without identifying patients at risk for early mortality. NEI-6 represents a novel tool to determine trauma activation appropriateness.

Associations Between Social Support and Social Media Use Among Young Adult Cisgender MSM and Transgender Women Living With HIV.

Social media platforms offer the opportunity to develop online social networks. Use of these platforms has been particularly attractive to younger sexual and gender minority individuals as well as those living with HIV. This cross-sectional study examined the perceived level of social support and associations with social media use among youth and young adult cisgender men who have sex with men (MSM) and transgender (trans) women living with HIV and examined these associations by gender identity. The study drew from baseline data collected from 612 cisgender MSM and 162 trans women enrolling in one of 10 demonstration sites that were part of a Health Resources and Services Administration Special Projects of National Significance initiative. The individual projects were designed to evaluate the potential for social media/mobile technology-based interventions to improve retention in care and HIV health outcomes. The data used in this study came from baseline surveys completed when participants enrolled in a site between October 2016 and May 2018. Results demonstrated that a significantly greater proportion of MSM than trans women participants reported the use of social media platforms (e.g., Facebook: MSM = 86%, trans women = 62%; Instagram: MSM = 65%, trans women = 35%). Furthermore, increased social media use improved perceptions of social support only among MSM participants (direct adjusted OR = 1.49) and not trans women participants (gender identity interaction term adjusted OR = 0.64). These results revealed that MSM participants perceived greater social benefit from the use of social media platforms than trans women, which could be a result of generalized online transphobia experienced by trans women. More nuanced data on various social media platforms, that is, anonymous versus profile-based, and group differences, are needed to better understand how social media platforms can be best utilized to optimize health care outcomes among sexual and gender minority youth and young adults living with HIV.

GNA13 loss in germinal center B cells leads to impaired apoptosis and promotes lymphoma in vivo.

GNA13 is the most frequently mutated gene in germinal center (GC)-derived B-cell lymphomas, including nearly a quarter of Burkitt lymphoma and GC-derived diffuse large B-cell lymphoma. These mutations occur in a pattern consistent with loss of function. We have modeled the GNA13-deficient state exclusively in GC B cells by crossing the Gna13 conditional knockout mouse strain with the GC-specific AID-Cre transgenic strain. AID-Cre(+) GNA13-deficient mice demonstrate disordered GC architecture and dark zone/light zone distribution in vivo, and demonstrate altered migration behavior, decreased levels of filamentous actin, and attenuated RhoA activity in vitro. We also found that GNA13-deficient mice have increased numbers of GC B cells that display impaired caspase-mediated cell death and increased frequency of somatic hypermutation in the immunoglobulin VH locus. Lastly, GNA13 deficiency, combined with conditional MYC transgene expression in mouse GC B cells, promotes lymphomagenesis. Thus, GNA13 loss is associated with GC B-cell persistence, in which impaired apoptosis and ongoing somatic hypermutation may lead to an increased risk of lymphoma development.

Targeting the Ras palmitoylation/depalmitoylation cycle in cancer.

The Ras proteins are well-known drivers of many cancers and thus represent attractive targets for the development of anticancer therapeutics. Inhibitors that disrupt the association of the Ras proteins with membranes by blocking the addition of the farnesyl lipid moiety to the Ras C-terminus failed in clinical trials. Here, we explore the possibility of targeting a second lipid modification, S-acylation, commonly referred to as palmitoylation, as a strategy to disrupt the membrane interaction of specific Ras isoforms. We review the enzymes involved in adding and removing palmitate from Ras and discuss their potential roles in regulating Ras tumorigenesis. In addition, we examine other proteins that affect Ras protein localization and may serve as future drug targets.

Genome-wide DNA methylation measurements in prostate tissues uncovers novel prostate cancer diagnostic biomarkers and transcription factor binding patterns.

BACKGROUND: Current diagnostic tools for prostate cancer lack specificity and sensitivity for detecting very early lesions. DNA methylation is a stable genomic modification that is detectable in peripheral patient fluids such as urine and blood plasma that could serve as a non-invasive diagnostic biomarker for prostate cancer. METHODS: We measured genome-wide DNA methylation patterns in 73 clinically annotated fresh-frozen prostate cancers and 63 benign-adjacent prostate tissues using the Illumina Infinium HumanMethylation450 BeadChip array. We overlaid the most significantly differentially methylated sites in the genome with transcription factor binding sites measured by the Encyclopedia of DNA Elements consortium. We used logistic regression and receiver operating characteristic curves to assess the performance of candidate diagnostic models. RESULTS: We identified methylation patterns that have a high predictive power for distinguishing malignant prostate tissue from benign-adjacent prostate tissue, and these methylation signatures were validated using data from The Cancer Genome Atlas Project. Furthermore, by overlaying ENCODE transcription factor binding data, we observed an enrichment of enhancer of zeste homolog 2 binding in gene regulatory regions with higher DNA methylation in malignant prostate tissues. CONCLUSIONS: DNA methylation patterns are greatly altered in prostate cancer tissue in comparison to benign-adjacent tissue. We have discovered patterns of DNA methylation marks that can distinguish prostate cancers with high specificity and sensitivity in multiple patient tissue cohorts, and we have identified transcription factors binding in these differentially methylated regions that may play important roles in prostate cancer development.

Resting Heart Rate Variability Among Professional Baseball Starting Pitchers.

Cornell, DJ, Paxson, JL, Caplinger, RA, Seligman, JR, Davis, NA, and Ebersole, KT. Resting heart rate variability among professional baseball starting pitchers. J Strength Cond Res 31(3): 575-581, 2017-The purpose of this study was to examine the changes in resting heart rate variability (HRV) across a 5-day pitching rotation schedule among professional baseball starting pitchers. The HRV data were collected daily among 8 Single-A level professional baseball starting pitchers (mean ± SD, age = 21.9 ± 1.3 years; height = 185.4 ± 3.6 cm; weight = 85.2 ± 7.5 kg) throughout the entire baseball season with the participant quietly lying supine for 10 minutes. The HRV was quantified by calculating the natural log of the square root of the mean sum of the squared differences (lnRMSSD) during the middle 5 minutes of each R-R series data file. A split-plot repeated-measures analysis of variance was used to examine the influence of pitching rotation day on resting lnRMSSD. A statistically significant main effect of rotation day was identified (F4,706 = 3.139, p = 0.029). Follow-up pairwise analyses indicated that resting lnRMSSD on day 2 was significantly (p ≤ 0.05) lower than all other rotation days. In addition, a statistically significant main effect of pitcher was also identified (F7,706 = 83.388, p < 0.001). These results suggest that professional baseball starting pitchers display altered autonomic nervous system function 1 day after completing a normally scheduled start, as day 2 resting HRV was significantly lower than all other rotation days. In addition, the season average resting lnRMSSD varied among participants, implying that single-subject analysis of resting measures of HRV may be more appropriate when monitoring cumulative workload among this cohort population of athletes.

In-Game Heart Rate Responses Among Professional Baseball Starting Pitchers.

Cornell, DJ, Paxson, JL, Caplinger, RA, Seligman, JR, Davis, NA, Flees, RJ, and Ebersole, KT. In-game heart rate responses among professional baseball starting pitchers. J Strength Cond Res 31(1): 24-29, 2017-The purpose of the current study was to characterize the in-game heart rate (HR) responses of baseball pitching. In-game HR was recorded from 16 professional baseball starting pitchers (mean ± SD, age = 22.1 ± 1.3 years; height = 187.9 ± 4.4 cm; weight = 90.5 ± 9.5 kg) for a total of 682 innings (home = 381, away = 301). All analyzed HR data were then normalized to each pitcher's age-predicted maximal HR (%HRmax). The group mean ± SD in-game %HRmax among all pitchers was 84.8 ± 3.9%, suggesting that baseball pitching is predominantly an anaerobic task. A split-plot mixed-model repeated measures analysis of variance identified a significant interaction effect between inning and game location (p = 0.042). Follow-up simple effects indicated that the in-game %HRmax was significantly different across innings, but only during home starts (p < 0.001). Specifically, pairwise analyses indicated that the in-game %HRmax during home starts were significantly (p ≤ 0.05) higher in the first and second innings than in all other innings. In addition, follow-up simple effects indicated that the in-game %HRmax was significantly (p = 0.017) higher during home starts than away starts in the first inning (87.3 ± 3.6% vs. 85.8 vs. 3.8%, respectively). Thus, it is possible that inning-dependent psychological factors may have contributed to the observed changes in in-game physiological intensity across innings and that these factors are specific to game location. Consequently, strength and conditioning practitioners should prescribe high-intensity exercises when developing conditioning programs for professional baseball starting pitchers.

Induced ablation of Bmp1 and Tll1 produces osteogenesis imperfecta in mice.

Osteogenesis imperfecta (OI), or brittle bone disease, is most often caused by dominant mutations in the collagen I genes COL1A1/COL1A2, whereas rarer recessive OI is often caused by mutations in genes encoding collagen I-interacting proteins. Recently, mutations in the gene for the proteinase bone morphogenetic 1 (BMP1) were reported in two recessive OI families. BMP1 and the closely related proteinase mammalian tolloid-like 1 (mTLL1) are co-expressed in various tissues, including bone, and have overlapping activities that include biosynthetic processing of procollagen precursors into mature collagen monomers. However, early lethality of Bmp1- and Tll1-null mice has precluded use of such models for careful study of in vivo roles of their protein products. Here we employ novel mouse strains with floxed Bmp1 and Tll1 alleles to induce postnatal, simultaneous ablation of the two genes, thus avoiding barriers of Bmp1(-/-) and Tll1(-/-) lethality and issues of functional redundancy. Bones of the conditionally null mice are dramatically weakened and brittle, with spontaneous fractures-defining features of OI. Additional skeletal features include osteomalacia, thinned/porous cortical bone, reduced processing of procollagen and dentin matrix protein 1, remarkably high bone turnover and defective osteocyte maturation that is accompanied by decreased expression of the osteocyte marker and Wnt-signaling inhibitor sclerostin, and by marked induction of canonical Wnt signaling. The novel animal model presented here provides new opportunities for in-depth analyses of in vivo roles of BMP1-like proteinases in bone and other tissues, and for their roles, and for possible therapeutic interventions, in OI.

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.

Transposase mediated construction of RNA-seq libraries.

RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequencing from double-stranded cDNA with only two enzymatic reactions. We generated high-quality RNA-seq libraries from as little as 10 pg of mRNA (∼1 ng of total RNA) with this approach. We also present a strand-specific RNA-seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. The directional RNA-seq libraries maintain the same quality as the nondirectional libraries, while showing a high degree of strand specificity, such that 99.5% of reads map to the expected genomic strand. Each transposon-based library construction method performed well when compared with standard RNA-seq library construction methods with regard to complexity of the libraries, correlation between biological replicates, and the percentage of reads that align to the genome as well as exons. Our results show that high-quality RNA-seq libraries can be constructed efficiently and in an automatable fashion using transposition technology.

Encore: Genetic Association Interaction Network centrality pipeline and application to SLE exome data.

Open source tools are needed to facilitate the construction, analysis, and visualization of gene-gene interaction networks for sequencing data. To address this need, we present Encore, an open source network analysis pipeline for genome-wide association studies and rare variant data. Encore constructs Genetic Association Interaction Networks or epistasis networks using two optional approaches: our previous information-theory method or a generalized linear model approach. Additionally, Encore includes multiple data filtering options, including Random Forest/Random Jungle for main effect enrichment and Evaporative Cooling and Relief-F filters for enrichment of interaction effects. Encore implements SNPrank network centrality for identifying susceptibility hubs (nodes containing a large amount of disease susceptibility information through the combination of multivariate main effects and multiple gene-gene interactions in the network), and it provides appropriate files for interactive visualization of a network using tools from our online Galaxy instance. We implemented these algorithms in C++ using OpenMP for shared-memory parallel analysis on a server or desktop. To demonstrate Encore's utility in analysis of genetic sequencing data, we present an analysis of exome resequencing data from healthy individuals and those with Systemic Lupus Erythematous (SLE). Our results verify the importance of the previously associated SLE genes HLA-DRB and NCF2, and these two genes had the highest gene-gene interaction degrees among the susceptibility hubs. An additional 14 genes previously associated with SLE emerged in our epistasis network model of the exome data, and three novel candidate genes, ST8SIA4, CMTM4, and C2CD4B, were implicated in the model. In summary, we present a comprehensive tool for epistasis network analysis and the first such analysis of exome data from a genetic study of SLE.

The salience network is responsible for switching between the default mode network and the central executive network: replication from DCM.

With the advent of new analysis methods in neuroimaging that involve independent component analysis (ICA) and dynamic causal modelling (DCM), investigations have focused on measuring both the activity and connectivity of specific brain networks. In this study we combined DCM with spatial ICA to investigate network switching in the brain. Using time courses determined by ICA in our dynamic causal models, we focused on the dynamics of switching between the default mode network (DMN), the network which is active when the brain is not engaging in a specific task, and the central executive network (CEN), which is active when the brain is engaging in a task requiring attention. Previous work using Granger causality methods has shown that regions of the brain which respond to the degree of subjective salience of a stimulus, the salience network, are responsible for switching between the DMN and the CEN (Sridharan et al., 2008). In this work we apply DCM to ICA time courses representing these networks in resting state data. In order to test the repeatability of our work we applied this to two independent datasets. This work confirms that the salience network drives the switching between default mode and central executive networks and that our novel technique is repeatable.

Recurrent read-through fusion transcripts in breast cancer.

Read-through fusion transcripts that result from the splicing of two adjacent genes in the same coding orientation are a recently discovered type of chimeric RNA. We sought to determine if read-through fusion transcripts exist in breast cancer. We performed paired-end RNA-seq of 168 breast samples, including 28 breast cancer cell lines, 42 triple negative breast cancer primary tumors, 42 estrogen receptor positive (ER+) breast cancer primary tumors, and 56 non-malignant breast tissue samples. We analyzed the sequencing data to identify breast cancer associated read-through fusion transcripts. We discovered two recurrent read-through fusion transcripts that were identified in breast cancer cell lines, confirmed across breast cancer primary tumors, and were not detected in normal tissues (SCNN1A-TNFRSF1A and CTSD-IFITM10). Both fusion transcripts use canonical splice sites to join the last splice donor of the 5' gene to the first splice acceptor of the 3' gene, creating an in-frame fusion transcript. Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells. Custom small interfering RNAs targeting the CTSD-IFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation. Read-through fusion transcripts between adjacent genes with different biochemical functions represent a new type of recurrent molecular defect in breast cancer that warrant further investigation as potential biomarkers and therapeutic targets. Both breast cancer associated fusion transcripts identified in this study involve membrane proteins (SCNN1A-TNFRSF1A and CTSD-IFITM10), which raises the possibility that they could be breast cancer-specific cell surface markers.

Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.

Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here a global characterization of rat neural palmitoyl-proteomes identifies most of the known neural palmitoyl proteins-68 in total, plus more than 200 new palmitoyl-protein candidates, with further testing confirming palmitoylation for 21 of these candidates. The new palmitoyl proteins include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is the finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl), both with regard to localization and function: Cdc42-palm concentrates in dendritic spines and has a special role in inducing these post-synaptic structures. Furthermore, assessing palmitoylation dynamics in drug-induced activity models identifies rapidly induced changes for Cdc42 as well as for other synaptic palmitoyl proteins, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.

Comprehensive mass spectrometric mapping of the hydroxylated amino acid residues of the α1(V) collagen chain.

BACKGROUND: α1(V) is an extensively modified collagen chain important in disease. RESULTS: Comprehensive mapping of α1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets. CONCLUSION: The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties. SIGNIFICANCE: Positions, numbers, and occupancy of modified sites can provide insights into α1(V) biological properties. Aberrant expression of the type V collagen α1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the α1(V) chain are linked to lung transplant rejection and atherosclerosis. The α1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the α1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental α1(V) and human recombinant pro-α1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these α1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed α1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of α1(V) chain and type V collagen properties.