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Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA-smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere–nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

Reliable models of renal failure in large animals are critical to the successful translation of the next generation of renal replacement therapies (RRT) into humans. While models exist for the induction of renal failure, none are optimized for the implantation of devices to the retroperitoneal vasculature. We successfully piloted an embolization-to-implantation protocol enabling the first implant of a silicon nanopore membrane hemodialyzer (SNMHD) in a swine renal failure model. Renal arterial embolization is a non-invasive approach to near-total nephrectomy that preserves retroperitoneal anatomy for device implants. Silicon nanopore membranes (SNM) are efficient blood-compatible membranes that enable novel approaches to RRT. Yucatan minipigs underwent staged bilateral renal arterial embolization to induce renal failure, managed by intermittent hemodialysis. A small-scale arteriovenous SNMHD prototype was implanted into the retroperitoneum. Dialysate catheters were tunneled externally for connection to a dialysate recirculation pump. SNMHD clearance was determined by intermittent sampling of recirculating dialysate. Creatinine and urea clearance through the SNMHD were 76–105 mL/min/m2 and 140–165 mL/min/m2, respectively, without albumin leakage. Normalized creatinine and urea clearance measured in the SNMHD may translate to a fully implantable clinical-scale device. This pilot study establishes a path toward therapeutic testing of the clinical-scale SNMHD and other implantable RRT devices.

The human body is composed of trillions of cells and exhibits remarkable biological complexity—far exceeding that of single-celled organisms like yeast or bacteria. Yet our genome contains only about 20,000 protein-coding genes, just a few times more than that of many bacteria. So where does this complexity arise? The answer lies not in the number of our genes, but in how we use them. Rather than producing a single protein from each gene, human cells employ multiple mechanisms to generate alternative, “hidden” protein variants—previously overlooked products of our own genome. Several molecular mechanisms enable this diversity. One well-established mechanism is alternative splicing, which produces multiple mRNA isoforms from a single gene, leading to distinct protein products. Another, more emerging mechanism involves alternative translation initiation, where the ribosome selects among multiple potential start codons on a single mRNA. Although translation canonically begins at an “AUG” codon, the ribosome can initiate at multiple start codons in a single mRNA—including non-AUG start codons—producing protein isoforms that share the same core sequence but differ at their N-termini. These alternative N-terminal isoforms—part of the “hidden proteome”—represent a vast and largely unexplored layer of biological complexity. Here, I combined high-throughput, molecular, and cell biological approaches to uncover the regulation, function, and physiological relevance of the hidden proteome. First, I characterized a specific alternative splicing event in a key cell division factor during meiosis and demonstrated its importance for fertility. I then discovered a global, cell cycle–dependent switch in alternative translation initiation. I show that this translational remodeling is mediated by the sequestration of the translation stringency factor eIF1 in the nucleus during interphase and its release into the cytoplasm during mitosis. Building on these findings, I demonstrated that alternative translation is a powerful and widespread mechanism for generating protein isoforms with distinct subcellular localizations. I further leveraged these insights to highlight the significance of alternative start codon selection in interpreting rare disease variants, introducing the concept of isoform-selective alleles. Finally, I expanded my analyses beyond alternative isoforms to investigate the roles of previously uncharacterized genes, identifying two novel factors essential for ribosome biogenesis and contributing to the systematic functional annotation of the genome. Together, these studies reveal fundamental principles governing the regulation and function of the “hidden proteome,” offering new perspectives on gene expression, cellular complexity, development, and human disease.

Regulated start codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms 1–3. However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling 4, we reveal a global increase in the stringency of start codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This enhanced stringency of start codon selection during mitosis results from increased associations between the 40S ribosome and the key regulator of start codon selection, eIF1. We find that increased eIF1–40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in the altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.

Regulated start-codon selection has the potential to reshape the proteome through the differential production of upstream open reading frames, canonical proteins, and alternative translational isoforms 1 – 3 . However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation-initiation-site profiling 4 , we reveal a global increase in the stringency of start-codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This enhanced stringency of start-codon selection during mitosis results from increased association between the 40S ribosome and the key regulator of start-codon selection, eIF1. We find that increased eIF1–40S ribosome interaction during mitosis is mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the change to translational stringency during mitosis, resulting in altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapies. Thus, cells globally control stringency of translation initiation, which has critical roles during the mammalian cell cycle in preserving mitotic cell physiology. Transcriptome-wide profiling studies in mammalian cells show that the stringency of start-codon selection is increased during mitosis, and that this is regulated by nuclear eIF1 to preserve mitotic arrest physiology.

Magnetic resonance neurography (MRN) has an increasing role in the diagnosis and management of pudendal neuralgia, a neurogenic cause of chronic pelvic pain. The objective of this research was to determine the role of MRN in predicting improved pain outcomes following computed tomography (CT)-guided perineural injections in patients with pudendal neuralgia. This study used a retrospective cross-sectional study design. The research was conducted at a large academic hospital. Patients: Ninety-one patients (139 injections) who received MRN and CT-guided pudendal blocks were analyzed. A 3Tesla (T) scanner was used to evaluate the lumbosacral plexus for pudendal neuropathy. Prior to receiving a CT-guided pudendal perineural injection, patients were given pain logs and asked to record pain on a visual analog scale. MRN findings for pudendal neuropathy were compared to the results of the CT-guided pudendal nerve blocks. Injection pain responses were categorized into 3 groups - positive block, possible positive block, and negative block.Statistical Tests: A chi-square test was used to test any association, and a Cochran-Armitage trend test was used to test any trend. Significance level was set at .05. All analyses were done in SAS Version 9.4 (SAS Institute, Inc., Cary, NC). Ninety-one patients (139 injections) who received MRN were analyzed. Of these 139 injections, 41 were considered positive (29.5%), 52 of 139 were possible positives (37.4%), and 46 of 139 were negative blocks (33.1%). Of the patients who had a positive pudendal block, no significant difference was found between the MRN result and the pudendal perineural injection response (P = .57). Women had better overall response to pudendal blocks, but this response was not associated with MRN findings (P = .34). However, positive MRN results were associated with better pain response in men (P = .005). Patients who reported bowel dysfunction also had a better response to pudendal perineural injection (P = .02). Some limitations include subjectivity of pain reporting, reporting consistency, absence of a control group, and the retrospective nature of the chart review. Pudendal perineural injections improve pain in patients with pudendal neuralgia and positive MRN results are associated with better response in men. MRI, MRN, CT injection, pudendal neuralgia, pudendal nerve, pelvic pain, chronic pelvic pain, pudendal neuropathy.

Miniaturized synthesis of positron emission tomography (PET) tracers is poised to offer numerous advantages including reduced tracer production costs and increased availability of diverse tracers. While many steps of the tracer production process have been miniaturized, there has been relatively little development of microscale systems for the quality control (QC) testing process that is required by regulatory agencies to ensure purity, identity, and biological safety of the radiotracer before use in human subjects. Every batch must be tested, and in contrast with ordinary pharmaceuticals, the whole set of tests of radiopharmaceuticals must be completed within a short-period of time to minimize losses due to radioactive decay. By replacing conventional techniques with microscale analytical ones, it may be possible to significantly reduce instrument cost, conserve lab space, shorten analysis times, and streamline this aspect of PET tracer production. We focus in this work on miniaturizing the subset of QC tests for chemical identity and purity. These tests generally require high-resolution chromatographic separation prior to detection to enable the approach to be applied to many different tracers (and their impurities), and have not yet, to the best of our knowledge, been tackled in microfluidic systems. Toward this end, we previously explored the feasibility of using the technique of capillary electrophoresis (CE) as a replacement for the “gold standard” approach of using high-performance liquid chromatography (HPLC) since CE offers similar separating power, flexibility, and sensitivity, but can readily be implemented in a microchip format. Using a conventional CE system, we previously demonstrated the successful separation of non-radioactive version of a clinical PET tracer, 3′-deoxy-3′-fluorothymidine (FLT), from its known by-products, and the separation of the PET tracer 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)-cytosine (D-FAC) from its α-isomer, with sensitivity nearly as good as HPLC. Building on this feasibility study, in this paper, we describe the first effort to miniaturize the chemical identity and purity tests by using microchip electrophoresis (MCE). The fully automated proof-of-concept system comprises a chip for sample injection, a separation capillary, and an optical detection chip. Using the same model compound (FLT and its known by-products), we demonstrate that samples can be injected, separated, and detected, and show the potential to match the performance of HPLC. Addition of a radiation detector in the future would enable analysis of radiochemical identity and purity in the same device. We envision that eventually this MCE method could be combined with other miniaturized QC tests into a compact integrated system for automated routine QC testing of radiopharmaceuticals in the future.Graphical abstractMiniaturized quality control (QC) testing of batches of radiopharmaceuticals via microfluidic analysis. The proof-of-concept hybrid microchip electrophoresis (MCE) device demonstrated the feasibility of achieving comparable performance to conventional analytical instruments (HPLC or CE) for chemical purity testing.

Production of a positron emission tomography (PET) tracer involves three stages: production of the radioisotope, radio-labeling and purification of the tracer, and quality control (QC) testing of the final product (if the tracer is used in humans). Though the production of the positron-emitting radioisotope requires expensive and complex instrumentation (e.g. cyclotron or generator), in many cases, the radioisotope can simply be obtained from a commercial source. The radio-labeling and purification step is performed by machines called radiosynthesizers, which perform chemical synthesis and purification processes in a remote-controlled or automated manner within radiation-shielded “hot cells”. Quality control testing is performed to ensure each batch of radiopharmaceutical is safe prior to use in human subjects, and requires several analytical chemistry instruments. Though a couple of PET tracers are routinely available in final form from commercial sources, most other tracers need to be specially manufactured and quality tested at the researcher’s facility. Advances in radiosynthesizer technology such as automation, programmability, and reagent kits simplify the production of diverse tracers. Furthermore, recent efforts in miniaturized synthesizers based on microfluidics aim to reduce equipment cost, shielding and lab space needs, and quantities of expensive reagents, to make it more affordable to produce custom batches of PET tracers on demand. In the context of these emerging technologies, quality control testing still remains a bottleneck due to the high cost of the many expensive instruments, specially-trained staff, and documentation needed to determine the purity and ensure the levels of all potential contaminants are below acceptable limits. Several companies are developing automated systems for quality control testing to alleviate some of these issues. However, high cost and large size remain obstacles. We have therefore explored the use of microfluidic analytical chemistry techniques to address these remaining critical issues. In this dissertation, the feasibility of using capillary electrophoresis (CE) to analyze the chemical purity of PET tracers is investigated as a compact and inexpensive replacement for high performance liquid chromatography (HPLC) and other techniques that are normally used for this purpose. After establishing that CE has sufficient performance (comparable to HPLC) using several example chemical systems, the design, development, and characterization of a microscale chemical purity testing system are presented, along with a discussion of its capabilities and limitations. Overall, it appears that microchip CE could be used instead of HPLC for performing many of the required QC tests of PET tracers.

Alternative splicing expands proteomic diversity and is tightly regulated by splicing factors, including the serine/arginine-rich (SR) protein family. Here, we analyze the poorly characterized protein SRSF12. Although SRSF12 is conserved across vertebrates, it is poorly expressed in most mammals, and we find that SRSF12 knockout mice do not display overt physiological or transcriptomic alterations. In contrast, SRSF12 is more highly expressed in primates where it is predominantly transcribed in the testes, oocytes, and brain. SRSF12 co-localizes with other splicing components to nuclear speckles and interacts with core splicing factors in cultured human cells. Strikingly, ectopic expression of SRSF12 in human cells induces widespread transcriptional changes, activating meiosis-, testis- and brain-specific genes. SRSF12 overexpression also leads to mitotic arrest and cell death, phenotypes that require both its structured RNA recognition motif and intrinsically disordered arginine/serine-rich C-terminal domain. Together, our results suggest that SRSF12 has evolved primate-specific expression to regulate testis- and brain-specific genes.

In the presence of cell division errors, mammalian cells can pause in mitosis for tens of hours with little to no transcription, while still requiring continued translation for viability. These unique aspects of mitosis require substantial adaptations to the core gene expression programs. Indeed, during interphase, the homeostatic control of mRNA levels involves a constant balance of transcription and degradation, with a median mRNA half-life of ~2-4 hours. If such short mRNA half-lives persisted in mitosis, cells would be expected to quickly deplete their transcriptome in the absence of new transcription. Here, we report that the transcriptome is globally stabilized during prolonged mitotic delays. Typical mRNA half-lives are increased >4-fold in mitosis compared to interphase, thereby buffering mRNA levels in the absence of new synthesis. Moreover, the poly(A)-tail-length profile of mRNAs changes in mitosis, strongly suggesting a mitotic repression of deadenylation. We further show that mRNA stabilization in mitosis is dependent on cytoplasmic poly(A)-binding proteins PABPC1&4. Depletion of PABPC1&4 disrupts the maintenance of mitotic arrest, highlighting the critical physiological role of mitotic transcriptome buffering.