Integrated compact regulators of protein activity enable control of signaling pathways and genome-editing in vivo.

Viral proteases and clinically safe inhibitors were employed to build integrated compact regulators of protein activity (iCROP) for post-translational regulation of functional proteins by tunable proteolytic activity. In the absence of inhibitor, the co-localized/fused protease cleaves a target peptide sequence introduced in an exposed loop of the protein of interest, irreversibly fragmenting the protein structure and destroying its functionality. We selected three proteases and demonstrated the versatility of the iCROP framework by validating it to regulate the functional activity of ten different proteins. iCROP switches can be delivered either as mRNA or DNA, and provide rapid actuation kinetics with large induction ratios, while remaining strongly suppressed in the off state without inhibitor. iCROPs for effectors of the NF-κB and NFAT signaling pathways were assembled and confirmed to enable precise activation/inhibition of downstream events in response to protease inhibitors. In lipopolysaccharide-treated mice, iCROP-sr-IκBα suppressed cytokine release ("cytokine storm") by rescuing the activity of IκBα, which suppresses NF-κB signaling. We also constructed compact inducible CRISPR-(d)Cas9 variants and showed that iCROP-Cas9-mediated knockout of the PCSK9 gene in the liver lowered blood LDL-cholesterol levels in mice. iCROP-based protein switches will facilitate protein-level regulation in basic research and translational applications.

Various Definitions of Failure Are Used in Studies of Patients Who Underwent Anterior Cruciate Ligament Reconstruction.

Purpose: To conduct a literature review to assess the definitions of anterior cruciate ligament reconstruction (ACLR) failure used throughout the orthopaedic literature. Methods: A systematic search of Embase, Ovid Medline, SPORTDiscus, and Web of Science was conducted by a university librarian to identity level I-IV clinical studies on ACLR failure. Inclusion criteria consisted of patients who underwent ACLR and included a definition of failure of ACLR. Patients who underwent anterior cruciate ligament (ACL) repairs, animal/cadaver studies, review studies, non-English language articles, and non-full text articles were excluded. Failure data were extracted from each study and categorized. Other data that were extracted included follow-up time after ACLR, failure reoperation rate, and failure reoperation procedure. Descriptive statistics was used to analyze the data. Results: Out of 2,775 studies, 104 (3.75%) met inclusion criteria and were analyzed in this review. The most common definition of ACLR failure included the use of a physical examination, specifically Lachman's test (21/104 [20.2%]), anterior laxity assessment, or a Pivot-Shift test (24/104 [35.2%]) or undergoing or requiring revision ACLR (39/104 [37.5%]). Although some studies used quantitative tests or imaging to help define "failure," others simply defined it as graft rerupture that was otherwise not defined (22/104 [22.5%]). Other common definitions included: the use of imaging (magnetic resonance imaging/radiographs) to confirm graft re-rupture (37/104 [35.6%]), patient-reported outcomes (recurrent instability)/patient reported outcomes measures (International Knee Documentation Committee [IKDC], Knee injury and Osteoarthritis Outcome Score [KOOS], Tegner) (18/104 [17.3%]), and the use of an arthrometer (KT-1000/2000, Rollimeter, or Kneelax) (17/104 [16.3%]). The least common definitions included graft failure or rerupture confirmed by arthroscopy (13/104 [12.5%]) and nonrevision surgery (2/104 [1.0%]). The failure rate of this procedure ranged from 0% to 100% depending on the definition of "failure." Conclusion: In this study, we found that a variety of definitions of failure are used among studies published in the orthopaedic literature. The most common criteria for failure of ACLR were the results of physical examination tests (35%), the need for undergoing a revision ACLR (36%), and the use of imaging to diagnose the failure (34%). About 17% of studies included in this review used patient-reported outcomes, specifically recurrent instability, or PROMs (IKDC, KOOS, Tegner) in their assessment of failure of ACLR. The least used definitions of "failure" of ACLR included nonrevision ACLR surgery (2%). Although some studies used similar tests or categories in their definition of failure, there were a variety of score and grade cutoff points between them. Level of Evidence: Level IV, systematic review of Level II-IV studies.

Rib Stress Fractures and a Novel Muscle-Sparing Rib Fixation Plating in an Elite Tennis Player: A Systematic Review of the Literature and Case Report.

A nationally ranked Division One female collegiate tennis player presented with pain in the chest and right posterior rib region after feeling a popping sensation during a routine overhead movement. The patient was eventually diagnosed with 2 lower rib stress fractures. After unsuccessful conservative management, the player underwent an open reduction and internal fixation and autologous bone grafting of the sixth and seventh ribs with a muscle-sparing approach and was able to return to full competition. We present this case along with a systematic review of the literature regarding rib stress fractures, which included 6 separate online data sources (PubMed, EMBASE, Cochrane, CINAHL, SportDiscus, and Medline). The successful surgical intervention demonstrates a significant option for elite athletes who wish to return to competition but have been unable to with standard of care conservative management of rib stress fractures.

Return to Sport After Primary Anterior Cruciate Ligament (ACL) Reconstruction: A Survey of The American Orthopaedic Society for Sports Medicine.

Introduction: Anterior cruciate ligament (ACL) tears are a common sports injury, and typically require a prolonged post-operative rehabilitation. The purpose of this study was to survey members of the American Orthopaedic Society for Sports Medicine (AOSSM) to determine their return to sport (RTS) criteria after primary ACL reconstruction (ACLR). Methods: A 23-question, anonymous survey hosted through Google® Docs was distributed electronically to AOSSM members. This survey included questions regarding the timing, as well as any functional tests or other metrics used to determine when an athlete is ready to RTS. Results: A total of 863 surgeons responded over four months. The most popular graft choice was bone patellar tendon bone autograft (63%). For non-pivoting sports, 43% of respondents allowed RTS at five to six months, while 31% allowed RTS at seven to eight months. For pivoting sports, 34% of respondents allowed RTS at seven to eight months, while 36% allowed RTS at nine to ten months. The most common criteria for return to non-pivoting sports include full knee motion (89%) and time after ACLR (76%). The most common criteria for return to pivoting sports include full knee motion (87%) and passing a hop test (80%). Only 21% of respondents assessed for psychological readiness to RTS. Conclusions: RTS occurred sooner in non-pivoting than pivoting sports, with similar RTS criteria in both groups. Most respondents did not assess for psychological readiness to RTS.

A Measure of Burnout in Current NCAA Student-Athletes.

Introduction: The prevalence of athletes who specialize in sports has increased in recent years. Substantial literature on youth sports has linked early sport specialization to negative consequences, such as burnout and injury. However, empirical evidence comparing rates of burnout and specialization in NCAA athletes is limited. The purpose of this study was to survey current NCAA Division I student-athletes to compare levels of burnout to sex, year of NCAA eligibility, and age at the beginning of sport specialization. Methods: A self-reported survey was distributed to student-athletes at two NCAA Division I institutions, which included demographics, sport specialization history, injury history, and the Athlete Burnout Questionnaire. Results from the three measures of the Athlete Burnout Questionnaire (reduced sense of accomplishment, exhaustion, sport devaluation) were compared to sex, year of NCAA eligibility, age of beginning sport specialization, and injury history. Results: A total of 267 athletes (95 males and 172 females) completed the survey. Of those, 156 (58.4%) were in their first or second year of NCAA eligibility, and 111 (41.6%) were in their third, fourth, or fifth year. Of the total, 121 (46.4%) reported specializing before the age of 15, and 140 (53.6%) specialized at age 16 or older. Females reported significantly higher levels of exhaustion than males (Difference of means (M) = 0.43, 95% confidence interval (CI) = [0.20, 0.66], p < 0.01). Athletes in their third, fourth, or fifth year of eligibility reported significantly higher levels of sport devaluation (M = 0.27, 95% CI = [0.05, 0.48], p < 0.05) than athletes in their first or second year. Athletes who specialized before age 15 did not report significantly higher levels of burnout than athletes who specialized at age 16 or later. In total, 203 athletes (77.2%) reported experiencing any injury. Athletes who reported a history of experiencing any injury demonstrated significantly higher feelings of reduced sense of accomplishment than athletes with no injury history (Difference of means (M) = 0.24, 95% confidence interval (CI) = [0.03, 0.45], p < 0.05). Conclusions: Athletes were more likely to experience elevated levels of burnout if they reported female sex, older NCAA eligibility, or a past injury history. However, athletes were not more likely to experience increased burnout based on age of beginning specialization. The results demonstrated a need to address burnout in athletes following injury and to be aware that females and older athletes are more prone to burnout.

Rapid Quantum Magnetic IL-6 Point-of-Care Assay in Patients Hospitalized with COVID-19.

Interleukin-6 (IL-6) has been linked to several life-threatening disease processes. Developing a point-of-care testing platform for the immediate and accurate detection of IL-6 concentrations could present a valuable tool for improving clinical management in patients with IL-6-mediated diseases. Drawing on an available biobank of samples from 35 patients hospitalized with COVID-19, a novel quantum-magnetic sensing platform is used to determine plasma IL-6 concentrations. A strong correlation was observed between IL-6 levels measured by QDTI10x and the Luminex assay (r = 0.70, p-value < 0.001) and between QDTI80x and Luminex (r = 0.82, p-value < 0.001). To validate the non-inferiority of QDTI to Luminex in terms of the accuracy of IL-6 measurement, two clinical parameters­the need for intensive care unit admission and the need for mechanical intubation­were chosen. IL-6 concentrations measured by the two assays were compared with respect to these clinical outcomes. Results demonstrated a comparative predictive performance between the two assays with a significant correlation coefficient. Conclusion: In short, the QDTI assay holds promise for implementation as a potential tool for rapid clinical decision in patients with IL-6-mediated diseases. It could also reduce healthcare costs and enable the development of future various biomolecule point-of-care tests for different clinical scenarios.

Differences in Outcomes Between Anterior and Posterior Shoulder Instability After Arthroscopic Bankart Repair: A Systematic Review and Meta-analysis.

BACKGROUND: The glenohumeral joint is one of the most frequently dislocated joints in the body, particularly in young, active adults. PURPOSE: To conduct a systematic review and meta-analysis to evaluate and compare outcomes between anterior versus posterior shoulder instability. STUDY DESIGN: Systematic review; Level of evidence, 4. METHODS: A systematic review was performed using the PubMed, Cochrane Library, and MEDLINE databases (from inception to September 2019) according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Studies were included if they were published in the English language, contained outcomes after anterior or posterior shoulder instability, had at least 1 year of follow-up, and included arthroscopic soft tissue labral repair of either anterior or posterior instability. Outcomes including return-to-sport (RTS) rate, postoperative instability rate, and pre- and postoperative American Shoulder and Elbow Surgeons (ASES) scores were recorded and analyzed. RESULTS: Overall, 39 studies were included (2077 patients; 1716 male patients and 361 female patients). Patients with anterior instability had a mean age of 23.45 ± 5.40 years (range, 11-72 years), while patients with posterior instability had a mean age of 23.08 ± 8.41 years (range, 13-61 years). The percentage of male patients with anterior instability was significantly higher than that of female patients (odds ratio [OR], 1.36; 95% CI, 1.04-1.77; P = .021). Compared with patients with posterior instability, those with anterior instability were significantly more likely to RTS (OR, 2.31; 95% CI, 1.76-3.04; P < .001), and they were significantly more likely to have postoperative instability (OR, 1.53; 95% CI, 1.07-2.23; P = .018). Patients with anterior instability also had significantly higher ASES scores than those with posterior instability (difference in means, 6.74; 95% CI, 4.71-8.77; P < .001). There were no significant differences found in postoperative complications between the anterior group (11 complications; 1.8%) and the posterior group (3 complications; 1.6%) (OR, 1.12; 95% CI, 0.29-6.30; P = .999). CONCLUSION: Patients with anterior shoulder instability had higher RTS rates but were more likely to have postoperative instability compared with posterior instability patients. Overall, male patients were significantly more likely to have anterior shoulder instability, while female patients were significantly more likely to have posterior shoulder instability.

Thyroid hormone receptor beta and NCOA4 regulate terminal erythrocyte differentiation.

An effect of thyroid hormone (TH) on erythropoiesis has been known for more than a century but the molecular mechanism(s) by which TH affects red cell formation is still elusive. Here we demonstrate an essential role of TH during terminal human erythroid cell differentiation; specific depletion of TH from the culture medium completely blocked terminal erythroid differentiation and enucleation. Treatment with TRß agonists stimulated premature erythroblast differentiation in vivo and alleviated anemic symptoms in a chronic anemia mouse model by regulating erythroid gene expression. To identify factors that cooperate with TRß during human erythroid terminal differentiation, we conducted RNA-seq in human reticulocytes and identified nuclear receptor coactivator 4 (NCOA4) as a critical regulator of terminal differentiation. Furthermore, Ncoa4-/- mice are anemic in perinatal periods and fail to respond to TH by enhanced erythropoiesis. Genome-wide analysis suggests that TH promotes NCOA4 recruitment to chromatin regions that are in proximity to Pol II and are highly associated with transcripts abundant during terminal differentiation. Collectively, our results reveal the molecular mechanism by which TH functions during red blood cell formation, results that are potentially useful to treat certain anemias.

Therapeutic genome editing: prospects and challenges.

Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease-based therapies as well as future prospects and challenges.

Blood biomarkers for brain injury in concussed professional ice hockey players.

IMPORTANCE: Lack of objective biomarkers for brain damage hampers acute diagnosis and clinical decision making about return to play after sports-related concussion. OBJECTIVES: To determine whether sports-related concussion is associated with elevated levels of blood biochemical markers of injury to the central nervous system and to assess whether plasma levels of these biomarkers predict return to play in professional ice hockey players with sports-related concussion. DESIGN, SETTING, AND PARTICIPANTS: Multicenter prospective cohort study involving all 12 teams of the top professional ice hockey league in Sweden, the Swedish Hockey League. Two hundred eighty-eight professional ice hockey players from 12 teams contesting during the 2012-2013 season consented to participate. All players underwent clinical preseason baseline testing regarding concussion assessment measures. Forty-seven players from 2 of the 12 ice hockey teams underwent blood sampling prior to the start of the season. Thirty-five players had a concussion from September 13, 2012, to January 31, 2013; of these players, 28 underwent repeated blood sampling at 1, 12, 36, and 144 hours and when the players returned to play. MAIN OUTCOMES AND MEASURES: Total tau, S-100 calcium-binding protein B, and neuron-specific enolase concentrations in plasma and serum were measured. RESULTS: Concussed players had increased levels of the axonal injury biomarker total tau(median, 10.0 pg/mL; range, 2.0-102 pg/mL) compared with preseason values (median, 4.5pg/mL; range, 0.06-22.7 pg/mL) (P < .001). The levels of the astroglial injury biomarker S-100 calcium-binding protein B were also increased in players with sports-related concussion(median, 0.075 µg/L; range, 0.037-0.24 µg/L) compared with preseason values (median,0.045 µg/L; range, 0.005-0.45 µg/L) (P < .001). The highest biomarker concentrations of total tau and S-100 calcium-binding protein B were measured immediately after a concussion, and they decreased during rehabilitation. No significant changes were detected in the levels of neuron-specific enolase from preseason values (median, 6.5 µg/L; range,3.45-18.0 µg/L) to postconcussion values (median, 6.1 µg/L; range, 3.6-12.8 µg/L) (P = .10). CONCLUSIONS AND RELEVANCE: Sports-related concussion in professional ice hockey players is associated with acute axonal and astroglial injury. This can be monitored using blood biomarkers, which may be developed into clinical tools to guide sport physicians in the medical counseling of athletes in return-to-play decisions.

Facet-sparing decompression with a minimally invasive flexible microblade shaver: a prospective operative analysis.

STUDY DESIGN: This is a detailed description of a facet-sparing decompression technique and a prospective observational study of 59 subjects. OBJECTIVE: To describe a facet-sparing decompression technique, quantify operative parameters, adverse events, and anatomic changes following decompression with a flexible microblade shaving system. SUMMARY OF BACKGROUND DATA: Decompression in patients with lumbar spinal stenosis is a common surgical procedure. However, obtaining a thorough decompression while leaving enough tissue to avoid destabilization can be challenging. Decompression with a flexible, through-the-foramen system may mitigate some of these challenges. MATERIALS AND METHODS: Fifty-nine subjects diagnosed with lumbar spinal stenosis were recruited into this study. Subjects underwent decompression with a flexible, microblade decompression system at a total of 88 levels between L2 and S1. Subject demographics, details of the procedure, and operation, including adverse events were collected. Preoperative and postoperative computed tomography scans and plain radiographs were obtained from a subset of 12 subjects and quantitatively assessed for bone removal and preservation of stabilizing structures. RESULTS: Fifty-nine subjects had 88 levels treated, 51% single-level and 49% 2-level with L4-L5 being the most commonly decompressed level. Operative time, blood loss, and length of stay were similar to or less than that seen in the historical control. The system was successfully used for decompression in 95.8% of the attempted foramina. Three operative complications were reported, all dural tears (5.1%). These dural tears occurred before introduction of the flexible decompression system. Computed tomography scans from 12 subjects demonstrate access to the lateral recess and foramen with removal of <6% of the superior facet cross-sectional area. CONCLUSIONS: The flexible microblade shaving system provided thorough decompression with few intraoperative complications. Operative variables were favorable compared to the literature and radiographic decompression was achieved to a great extent while allowing for the preservation of the facet joints and midline structures.

Olympic boxing is associated with elevated levels of the neuronal protein tau in plasma.

OBJECTIVES: The aim of this study was to investigate if olympic (amateur) boxing is associated with elevation of brain injury biomarkers in peripheral blood compared to controls. MATERIALS AND METHODS: Thirty olympic boxers competing in at least 47 bouts were compared to 25 controls. Blood was collected from the controls at one occasion and from the boxers within 1-6 days after a bout and after a rest period of at least 14 days. Tau concentration in plasma was determined using a novel single molecule ELISA assay and S-100B, glial fibrillary acidic protein, brain-derived neurotrophic factor and amyloid ß 1-42 were determined using standard immunoassays. RESULTS: None of the boxers had been knocked-out during the bout. Plasma-tau was significantly increased in the boxers after a bout compared to controls (mean ± SD, 2.46 ± 5.10 vs. 0.79 ± 0.961 ng L(-1), p = 0.038). The other brain injury markers did not differ between the groups. Plasma-tau decreased significantly in the boxers after a resting period compared to after a bout (p = 0.030). CONCLUSIONS: Olympic boxing is associated with elevation of tau in plasma. The repetitive minimal head injury in boxing may lead to axonal injuries that can be diagnosed with a blood test.

Tau proteins in serum predict neurological outcome after hypoxic brain injury from cardiac arrest: results of a pilot study.

OBJECTIVE: To conduct a pilot study to evaluate the prognostic potential of serum tau protein measurements to predict neurological outcome 6 months following resuscitation from cardiac arrest. METHODS: In this retrospective observational study, we employed a new ultra sensitive digital immunoassay technology to examine serial serum samples from 25 cardiac arrest patients to examine tau release into serum as a result of brain hypoxia, and probe for its significance predicting six-month neurological outcome. Serial blood samples were obtained from resuscitated cardiac arrest survivors during their first five days in an intensive care unit, and serum total tau was measured. Cerebral function assessments were made using Cerebral Performance Categorization (CPC) at discharge from the ICU and six months later. Tau data were analyzed in the context of 6-month CPC scores. RESULTS: Tau elevations ranged from modest (<10 pg/mL) to very high (hundreds of pg/mL), and exhibited unexpected bi-modal kinetics in some patients. Early tau elevations appeared within 24h of cardiac arrest, and delayed elevations appeared after 24-48 h. In patients with delayed elevations, areas under the curves of tau concentration vs. hours since cardiac arrest were highly predictive of 6-month outcome (P<0.0005). CONCLUSION: High-sensitivity serum tau measurements combined with an understanding of tau release kinetics could have utility for hypoxic brain injury assessment and prediction of cerebral function outcome.

Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.

The ability to detect single protein molecules in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as approximately 10-20 enzyme-labeled complexes in 100 microl of sample (approximately 10(-19) M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10(-15) M) much lower than conventional ELISA. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).

Rapid quantitative analysis using a single molecule counting approach.

Single molecule detection of target molecules specifically bound by paired fluorescently labeled probes has shown great potential for sensitive quantitation of biomolecules. To date, no reports have rigorously evaluated the analytical capabilities of a single molecule detection platform employing this dual-probe approach or the performance of its data analysis methodology. In this paper, we describe a rapid, automated, and sensitive multicolor single molecule detection apparatus and a novel extension of coincident event counting based on detection of fluorescent probes. The approach estimates the number of dual-labeled molecules of interest from the total number of coincident fluorescent events observed by correcting for unbound probes that randomly pass through the interrogation zone simultaneously. Event counting was evaluated on three combinations of distinct fluorescence channels and was demonstrated to outperform conventional spatial cross-correlation in generating a wider linear dynamic response to target molecules. Furthermore, this approach succeeded in detecting subpicomolar concentrations of a model RNA target to which fluorescently labeled oligonucleotide probes were hybridized in a complex background of RNA. These results illustrate that the fluorescent event counting approach described represents a general tool for rapid sensitive quantitative analysis of any sample analyte, including nucleic acids and proteins, for which pairs of specific probes can be developed.

Endogenous ouabain: upregulation of steroidogenic genes in hypertensive hypothalamus but not adrenal.

BACKGROUND: Mammalian tissues contain a presumed endogenous Na+, K(+)-ATPase inhibitor that binds reversibly to the Na+ pump with high affinity and specificity. The inhibitor has been linked to the pathogenesis of experimental volume-expanded and human essential hypertension. This compound has been isolated from mammalian hypothalamus and appears to be an isomer of the plant-derived cardiac glycoside ouabain, if not ouabain itself. The objective of this study was to test the hypothesis that a biosynthetic pathway exists in mammalian tissues to produce a steroid derivative closely related to plant cardiac glycosides. METHODS AND RESULTS: Using bioinformatics and genomic techniques, Milan hypertensive rat tissues were studied because this strain has a 10-fold increase in hypothalamic ouabain-like compound that is linked to the pathogenesis of the hypertension. A putative steroid biosynthetic pathway was constructed and candidate genes encoding enzymes in this pathway were identified from sequence databases. Differential expression of selected genes in the pathway was studied by microarray analysis and quantitative polymerase chain reaction, with functional validation by gene silencing using small interfering RNAs. Marked upregulation of genes coding for P450 side chain cleavage and Delta5-3beta-hydroxysteroid dehydrogenase/Delta5-Delta4- isomerase enzymes in hypertensive hypothalamus but not adrenal was found, compared with normotensive Milan rats. Knockdown of the latter gene decreased production of ouabain-like factor from neural tissue. CONCLUSIONS: Our findings support the possibility that a unique steroid biosynthetic circuit exists in Milan rat brain, functioning independently from adrenal, which could account for the overproduction of the hypothalamic ouabain-like compound in this species.

Role of the fetal and alpha/beta exons in the function of fast skeletal troponin T isoforms: correlation with altered Ca2+ regulation associated with development.

In mammalian fast skeletal muscle, constitutive and alternative splicing from a single troponin T (TnT) gene produce multiple developmentally regulated and tissue specific TnT isoforms. Two exons, alpha (exon 16) and beta (exon 17), located near the 3' end of the gene and coding for two different 14 amino acid residue peptides are spliced in a mutually exclusive manner giving rise to the adult TnTalpha and the fetal TnTbeta isoforms. In addition, an acidic peptide coded by a fetal (f) exon located between exons 8 and 9 near the 5' end of the gene, is specifically present in TnTbeta and absent in the adult isoforms. To define the functional role of the f and alpha/beta exons, we constructed combinations of TnT cDNAs from a single human fetal fast skeletal TnTbeta cDNA clone in order to circumvent the problem of N-terminal sequence heterogeneity present in wild-type TnT isoforms, irrespective of the stage of development. Nucleotide sequences of these constructs, viz. TnTalpha, TnTalpha + f, TnTbeta - f and TnTbeta are identical, except for the presence or absence of the alpha or beta and f exons. Our results, using the recombinant TnT isoforms in different functional in vitro assays, show that the presence of the f peptide in the N-terminal T1 region of TnT, has a strong inhibitory effect on binary interactions between TnT and other thin filament proteins, TnI, TnC and Tm. The presence of the f peptide led to reduced Ca2+-dependent ATPase activity in a reconstituted thin filament, whereas the contribution of the alpha and beta peptides in the biological activity of TnT was primarily modulatory. These results indicate that the f peptide confers an inhibitory effect on the biological function of fast skeletal TnT and this can be correlated with changes in the Ca2+ regulation associated with development in fast skeletal muscle.

Regulation of expression of the stress response gene, Osp94: identification of the tonicity response element and intracellular signalling pathways.

Osp94 (osmotic stress protein of 94 kDa) is known to be up-regulated by hypertonic and heat-shock stresses in mouse renal inner medullary collecting duct (mIMCD3) cells. To investigate the molecular mechanism of transcriptional regulation of the Osp94 gene under these stresses, we cloned and characterized the 5'-flanking region of the gene. Sequence analysis of the proximal 4 kb 5'-flanking region revealed a TATA-less G/C-rich promoter region containing a cluster of Sp1 sites. We also identified upstream sequence motifs similar to the consensus TonE/ORE (tonicity-response element/osmotic response element) as well as the consensus HSE (heat-shock element). Luciferase activities in cells transfected with reporter constructs containing a TonE/ORE-like element (Osp94-TonE; 5'-TGGAAAGGACCAG-3') and HSE enhanced reporter gene expression under hypertonic stress and heat-shock stress respectively. Electrophoretic gel mobility-shift assay showed a slowly migrating band binding to the Osp94-TonE probe, probably representing binding of TonEBP (TonE binding protein) to this enhancer element. Furthermore, treatment of mIMCD3 cells with MAPK (mitogen-activated protein kinase) inhibitors (SB203580, PD98059, U0126 and SP600125) and a proteasome inhibitor (MG132) suppressed the increase in Osp94 gene expression caused by hypertonic NaCl. These results indicate that the 5'-flanking region of Osp94 gene contains a hypertonicity sensitive cis -acting element, Osp94-TonE, which is distinct from a functional HSE. Furthermore, the MAPK and proteasome systems appear to be, at least in part, involved in hypertonic-stressmediated regulation of Osp94 through Osp94-TonE.

Drug discovery targeted to the Alzheimer's APP mRNA 5'-untranslated region: the action of paroxetine and dimercaptopropanol.

We screened for drugs that specifically interact with the 5'-untranslated region of the mRNA encoding the Alzheimer's amyloid precursor protein (APP). Our goal was to use newly discovered APP 5' UTR directed compounds to limit amyloid-beta (Abeta)-peptide output in cell culture systems. The APP 5' UTR folds into a stable RNA secondary structure (Gibbs free energy: DeltaG = -54.9 kcal/mol) and is an important regulator of the amount of APP translated in response to IL-1 (Nilsson et al., 1998; Rogers et al., 1999) and iron (Rogers et al., 2002). Seventeen drug "hits" were identified from a library of 1,200 FDA preapproved drugs (Rogers et al., 2002). Six of the original 17 compounds were validated for their capacity to suppress reporter gene expression in stable neuroblastoma transfectants expressing the dicistronic reporter construct shown in Fig. 2. These six leads suppressed APP 5' UTR driven luciferase translation while causing no effect on the translation of dicistronic GFP gene translated from a viral IRES (negative control to ensure specificity during drug screens). In this report, we show that paroxetine (serotonin reuptake blocker) and dimercaptopropanol (Hg chelator) exerted significant effects on APP expression (steady-state levels of APP), whereas Azithromycin altered APP processing. None of these three compounds altered APLP-1 expression. In the future, we will identify further novel compounds that influence Abeta levels, either via translation inhibition or by changing the activity of proteins coupled between APP translation and APP processing.