Tools and methods for high-throughput single-cell imaging with the mother machine.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning-based segmentation, 'what you put is what you get' (WYPIWYG) - that is, pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother machine-based high-throughput imaging and analysis methods in their research.

Enricherator: A Bayesian Method for Inferring Regularized Genome-wide Enrichments from Sequencing Count Data.

A pervasive question in biological research studying gene regulation, chromatin structure, or genomics is where, and to what extent, does a signal of interest arise genome-wide? This question is addressed using a variety of methods relying on high-throughput sequencing data as their final output, including ChIP-seq for protein-DNA interactions,1 GapR-seq for measuring supercoiling,2 and HBD-seq or DRIP-seq for R-loop positioning.3,4 Current computational methods to calculate genome-wide enrichment of the signal of interest usually do not properly handle the count-based nature of sequencing data, they often do not make use of the local correlation structure of sequencing data, and they do not apply any regularization of enrichment estimates. This can result in unrealistic estimates of the true underlying biological enrichment of interest, unrealistically low estimates of confidence in point estimates of enrichment (or no estimates of confidence at all), unrealistic gyrations in enrichment estimates at very close (<10 bp) genomic loci due to noise inherent in sequencing data, and in a multiple-hypothesis testing problem during interpretation of genome-wide enrichment estimates. We developed a tool called Enricherator to infer genome-wide enrichments from sequencing count data. Enricherator uses the variational Bayes algorithm to fit a generalized linear model to sequencing count data and to sample from the approximate posterior distribution of enrichment estimates (https://github.com/jwschroeder3/enricherator). Enrichments inferred by Enricherator more precisely identify known binding sites in cases where low coverage between binding sites leads to false-positive peak calls in these noisy regions of the genome; these benefits extend to published datasets.

Tools and methods for high-throughput single-cell imaging with the mother machine.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely-used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning based segmentation, "what you put is what you get" (WYPIWYG) - i.e., pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother-machine-based high-throughput imaging and analysis methods in their research.

Deep mutational scanning highlights a role for cytosolic regions in Hrd1 function.

Misfolded endoplasmic reticulum (ER) proteins are degraded through a process called ER-associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identify several regions required for different Hrd1 functions. Most surprisingly, we find two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we define roles for disordered regions between structural elements that are required for Hrd1 autoubiquitination and substrate interaction. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the ER membrane.

RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome wide.

RNA:DNA hybrids compromise replication fork progression and genome integrity in all cells. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate strongly in noncoding RNAs and 5'-UTRs of coding sequences. For ΔrnhB, hybrids accumulate preferentially in untranslated regions and early in coding sequences. We show that hybrid accumulation is particularly sensitive to gene expression in ΔrnhC cells. DNA replication in ΔrnhC cells is disrupted, leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts cause mutagenesis and shape genome organization.

RNase H genes cause distinct impacts on RNA:DNA hybrid formation and mutagenesis genome-wide.

RNA:DNA hybrids such as R-loops affect genome integrity and DNA replication fork progression. The overall impacts of naturally occurring RNA:DNA hybrids on genome integrity, and the relative contributions of ribonucleases H to mitigating the negative effects of hybrids, remain unknown. Here, we investigate the contributions of RNases HII (RnhB) and HIII (RnhC) to hybrid removal, DNA replication, and mutagenesis genome-wide. Deletion of either rnhB or rnhC triggers RNA:DNA hybrid accumulation, but with distinct patterns of mutagenesis and hybrid accumulation. Across all cells, hybrids accumulate most strongly in non-coding RNAs and 5'-UTRs of coding sequences. For Δ rnhB , hybrids accumulate preferentially in untranslated regions and early in coding sequences. Hybrid accumulation is particularly sensitive to gene expression in Δ rnhC ; in cells lacking RnhC, DNA replication is disrupted leading to transversions and structural variation. Our results resolve the outstanding question of how hybrids in native genomic contexts interact with replication to cause mutagenesis and shape genome organization.

Deep mutational scanning highlights a new role for cytosolic regions in Hrd1 function.

Misfolded endoplasmic reticulum proteins are degraded through a process called endoplasmic reticulum associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identified several regions required for different Hrd1 functions. Most surprisingly, we found two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we defined roles for disordered regions between structural elements that were required for Hrd1's ability to autoubiquitinate and interact with substrate. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the endoplasmic reticulum membrane.

Midsubstance Vastus Lateralis Tear in the Young Athlete: Case Report and a Review of the Literature.

ABSTRACT: Vastus lateralis tendon tear is an infrequent cause of lateral knee pain. Previously reported cases have described acute injury in middle-aged men after eccentric quadriceps contraction. This case report discusses 2 adolescent patients with longitudinal midsubstance tears diagnosed with MRI and dynamic ultrasound and treated successfully with operative intervention.

Low-volume Hydrodissection for the Treatment of Chronic Achilles Tendinopathy.

Chronic Achilles tendinopathy (AT) is a common ailment for many active duty service members that adversely affects readiness. Patients present with pain, swelling, and limited functional ability. Kager's fat pad is a mass of adipose tissue that protects the blood vessels supplying the Achilles tendon and preserves its function. A popular hypothesis is that scarring, tethering, and neovascularization play a significant role in the pathogenesis of AT. Current literature supports the effectiveness of high-volume (40-50 mL) hydrodissection, a procedure in which fluid is injected under ultrasound guidance into the tissues surrounding the Achilles tendon to mechanically separate the paratenon from the underlying Kager's fat pad. There may also be a beneficial effect of scar tissue and neoneurovascular breakdown. However, high-volume injections result in short-term discomfort and decreased mobility. Lowering injection volume (2-10 mL) may reduce this morbidity and facilitate use in limited-resource environments. This case report presents a 29-year-old active duty male with recalcitrant post-traumatic AT who achieved significant pain reduction and faster return to full service using low-volume hydrodissection. The use of 10 mL volume has not been described previously and provides additional support for using lower volumes in chronic AT. This technique is a direct adjunctive treatment option with rehabilitation at a military treatment facility or in the operational environment.

Hip Fractures: Diagnosis and Management.

Hip fractures are common causes of disability, with mortality rates reaching 30% at one year. Nonmodifiable risk factors include lower socioeconomic status, older age, female sex, prior fracture, metabolic bone disease, and bony malignancy. Modifiable risk factors include low body mass index, having osteoporosis, increased fall risk, medications that increase fall risk or decrease bone mineral density, and substance use. Hip fractures present with anterior groin pain, inability to bear weight, or a shortened, abducted, externally rotated limb. Plain radiography is usually sufficient for diagnosis, but magnetic resonance imaging should be obtained if suspicion of fracture persists despite normal radiography. Operative management within 24 to 48 hours of the fracture optimizes outcomes. Fractures are usually managed by surgery, with the approach based on fracture type and location; spinal or general anesthesia can be used. Nonsurgical management can be considered for patients who are not good surgical candidates. Pre- and postoperative antistaphylococcal antibiotics are given to prevent joint infection. Medications for venous thromboembolism prophylaxis are also recommended. Physicians should be alert for the presence of delirium, which is a common postoperative complication. Early postoperative mobilization, followed by rehabilitation, improves outcomes. Subsequent care focuses on prevention, with increased physical activity, home safety assessments, and minimizing polypharmacy. Two less common hip fractures can also occur: femoral neck stress fractures and insufficiency fractures. Femoral neck stress fractures typically occur in dancers 20 to 30 years of age, endurance athletes, and military service members, often because of training overload. Insufficiency fractures due to compromised bone strength occur without trauma in postmenopausal women. If not recognized and treated, these fractures can progress to complete and displaced fractures with high rates of nonunion and avascular necrosis.

A Population-Level Summary of Health Care Utilization for the Management of Patellar Tendinopathy in the Military Health System.

Patellar tendinopathy (PT) is a common nontraumatic orthopaedic disorder of the knee suffered by many service members. Understanding the make-up of usual care for PT at the system level can better frame current clinical gaps and areas that need improvement. Exercise therapy is recommended as a core treatment for PT, but it is unclear how often it is used as a part of usual care for PT within the Military Health System (MHS). The purpose of the study was to identify interventions used in the management of PT and the timing of these interventions. A secondary purpose was to determine if exercise therapy use was associated with reduced recurrence of knee pain. In total, 4,719 individuals aged 17 to 50 years in the MHS diagnosed with PT between 2010 and 2011 were included. Pharmacological and nonpharmacological interventions, visits to specialty providers, and imaging services were captured. Descriptive statistics were used to report the findings. Interventions were further categorized as being part of initial care (within the first 7 days), the initial episode of care (within the first 60 days), or the 2-year time period after diagnosis. Linear regression assessed the relationship between the number of exercise therapy visits in the initial episode of care and recurrences of knee pain. In total, 50.6% of this cohort had no more than one medical visit total for PT. Exercise therapy (18.2%) and nonsteroidal anti-inflammatory drugs (4.3%) were the two most used interventions in the initial episode of care. Radiographs were ordered for 23.1% of the cohort in the initial episode of care. The number of exercise therapy visits a patient received during the initial episode of care was not associated with recurrences of knee pain. Half of the individuals received no further care beyond an initial visit for the diagnosis of PT. Exercise therapy was the most common intervention used during the initial episode of care, but exercise therapy did not influence the recurrence of knee pain.

The nucleotide messenger (p)ppGpp is an anti-inducer of the purine synthesis transcription regulator PurR in Bacillus.

The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here, we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis gene expression upon amino acid starvation. We first identified PurR as a receptor of (p)ppGpp in Bacillus anthracis. A co-structure with Bacillus subtilis PurR reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of phosphoribosyltransferase enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant in B. subtilis fails to downregulate purine synthesis genes upon amino acid starvation. Our work establishes the precedent of (p)ppGpp as an effector of a classical transcription repressor and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from soil bacteria to pathogens.

Athlete Preparticipation Physical Evaluation.

Preparticipation evaluations (PPE) are both a traditional and legal requirement by many governing bodies for sport. The ideal goal of the PPE is safe participation in sport for athletes. This article provides an overview of common PPE elements and current best practice recommendations. Descriptions of every possible examination are published elsewhere and are beyond the intent of this article. Additional considerations for transgender, masters athletes, and Special Olympians are also outside the scope of this review, but are well covered in The Preparticipation Physical Evaluation, fifth edition monograph.

Nucleoid-associated proteins shape chromatin structure and transcriptional regulation across the bacterial kingdom.

Genome architecture has proven to be critical in determining gene regulation across almost all domains of life. While many of the key components and mechanisms of eukaryotic genome organization have been described, the interplay between bacterial DNA organization and gene regulation is only now being fully appreciated. An increasing pool of evidence has demonstrated that the bacterial chromosome can reasonably be thought of as chromatin, and that bacterial chromosomes contain transcriptionally silent and transcriptionally active regions analogous to heterochromatin and euchromatin, respectively. The roles played by histones in eukaryotic systems appear to be shared across a range of nucleoid-associated proteins (NAPs) in bacteria, which function to compact, structure, and regulate large portions of bacterial chromosomes. The broad range of extant NAPs, and the extent to which they differ from species to species, has raised additional challenges in identifying and characterizing their roles in all but a handful of model bacteria. Here we review the regulatory roles played by NAPs in several well-studied bacteria and use the resulting state of knowledge to provide a working definition for NAPs, based on their function, binding pattern, and expression levels. We present a screening procedure which can be applied to any species for which transcriptomic data are available. Finally, we note that NAPs tend to play two major regulatory roles - xenogeneic silencers and developmental regulators - and that many unrecognized potential NAPs exist in each bacterial species examined.

Femoroacetabular Impingement Syndrome.

Femoroacetabular impingement (FAI) syndrome is one of the most rapidly evolving etiologies of hip pain. The 2016 Warwick Agreement consensus statement defined FAI syndrome as a triad of symptoms, signs, and radiographic findings. Cam morphology is more likely in athletes and is associated with repetitive hip loading in maximal flexion during adolescence. Much less is known about the development of pincer morphology. Physical therapy improves pain and function, justifying a trial before pursuing surgery. Musculoskeletal injections are utilized for FAI syndrome, but the evidence is limited. Arthroscopic surgery for FAI syndrome can correct the morphological changes and address the underlying soft tissue injuries. Recent studies evaluated reliable indicators of surgical outcomes, the most reliable of which is the presurgical presence of osteoarthritis. Recent studies demonstrate the efficacy of surgery, but with the risk of complication and no guarantee of a return to the same level of sport.

The roles of replication-transcription conflict in mutagenesis and evolution of genome organization.

Replication-transcription conflicts promote mutagenesis and give rise to evolutionary signatures, with fundamental importance to genome stability ranging from bacteria to metastatic cancer cells. This review focuses on the interplay between replication-transcription conflicts and the evolution of gene directionality. In most bacteria, the majority of genes are encoded on the leading strand of replication such that their transcription is co-directional with the direction of DNA replication fork movement. This gene strand bias arises primarily due to negative selection against deleterious consequences of head-on replication-transcription conflict. However, many genes remain head-on. Can head-on orientation provide some benefit? We combine insights from both mechanistic and evolutionary studies, review published work, and analyze gene expression data to evaluate an emerging model that head-on genes are temporal targets for adaptive mutagenesis during stress. We highlight the alternative explanation that genes in the head-on orientation may simply be the result of genomic inversions and relaxed selection acting on nonessential genes. We seek to clarify how the mechanisms of replication-transcription conflict, in concert with other mutagenic mechanisms, balanced by natural selection, have shaped bacterial genome evolution.

Methyltransferase DnmA is responsible for genome-wide N6-methyladenosine modifications at non-palindromic recognition sites in Bacillus subtilis.

The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.

Metabolic Remodeling during Biofilm Development of Bacillus subtilis.

Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology.IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

Discovery of a dual protease mechanism that promotes DNA damage checkpoint recovery.

The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells promote checkpoint recovery after sensing successful repair is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. We show that DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that both proteases interact with YneA and that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.