Design and Synthesis of Novel Squaraine-Based Fluorescent Probe for Far-Red Detection of Chymotrypsin Enzyme.

Chymotrypsin, a crucial enzyme in human digestion, catalyzes the breakdown of milk proteins, underscoring its significance in both health diagnostics and dairy quality assurance. Addressing the critical need for rapid, cost-effective detection methods, we introduce a groundbreaking approach utilizing far-red technology and HOMO-Förster resonance energy transfer (FRET). Our novel probe, SQ-122 PC, features a unique molecular design that includes a squaraine dye (SQ), a peptide linker, and SQ moieties synthesized through solid-phase peptide synthesis. Demonstrating a remarkable quenching efficiency of 93.75% in a tailored H2O:DMSO (7:3) solvent system, our probe exhibits absorption and emission properties within the far-red spectrum, with an unprecedented detection limit of 0.130 nM. Importantly, our method offers unparalleled selectivity towards chymotrypsin, ensuring robust and accurate enzyme detection. This pioneering work underscores the immense potential of far-red-based homo-FRET systems in enabling the sensitive and specific detection of chymotrypsin enzyme activity. By bridging the gap between cutting-edge technology and biomedical diagnostics, our findings herald a new era of enzyme sensing, promising transformative advancements in disease diagnosis and dairy quality control.

Photophysical Characterization and Biointeractions of NIR Squaraine Dyes for in Vitro and in Vivo Bioimaging.

The increasing demand for reliable near-infrared (NIR) probes exhibiting enduring fluorescence in living systems and facile compatibility with biomolecules such as peptides, antibodies or proteins is driven by the increasing use of NIR imaging in clinical diagnostics. To address this demand, a series of carboxy-functionalized unsymmetrical squaraine dyes (SQ-27, SQ-212, and SQ-215) along with non-carboxy-functionalized SQ-218 absorbing and emitting in the NIR wavelength range were designed and synthesized followed by photophysical characterization. This study focused on the impact of structural variations in the alkyl chain length, carboxy functionality positioning, and spacer chain length on dye aggregation and interaction with bovine serum albumin (BSA) as a model protein. In phosphate buffer (PB), the absorption intensity of the dyes markedly decreased accompanied by pronounced shoulders indicative of dye aggregation, and complete fluorescence quenching was seen in contrast to organic solvents. However, in the presence of BSA in PB, there was a enhancement in absorption intensity while regaining the fluorescence coupled with a remarkable increase in the intensity with increasing BSA concentrations, signifying the impact of dye-BSA interactions on preventing aggregation. Further analysis of Job's plot unveiled a 2:1 interaction ratio between BSA and all dyes, while the binding studies revealed a robust binding affinity (Ka) in the order of 107/mol. SQ-212 and SQ-215 were further tested for their in vitro and in vivo imaging capabilities. Notably, SQ-212 demonstrated nonpermeability to cells, while SQ-215 exhibited easy penetration and prominent cytoplasmic localization in in vitro studies. Injection of the dyes into laboratory mice showcased their efficacy in visualization, displaying stable and intense fluorescence in tissues without toxicity, organ damage, or behavioral changes. Thus, SQ-212 and SQ-215 are promising candidates for imaging applications, holding potential for noninvasive cellular and diagnostic imaging as well as biomarker detection when coupled with specific vectors in living systems.

CGG repeat expansion in LRP12 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.

In Vitro and In Silico Studies on Angiotensin I-Converting Enzyme Inhibitory Peptides Found in Hydrophobic Domains of Porcine Elastin.

One of the most striking aspects of the primary structure in the hydrophobic domains of the tropoelastin molecule is the occurrence of the VAPGVG repeating sequence. Since the N-terminal tripeptide VAP of VAPGVG showed a potent ACE inhibitory activity, the ACE inhibitory activity of various derivatives of VAP was examined in vitro. The results showed that VAP derivative peptides VLP, VGP, VSP, GAP, LSP, and TRP exhibited potent ACE inhibitory activities, while the non-derivative peptide APG showed only weak activity. In in silico studies, the docking score S value showed that VAP derivative peptides VLP, VGP, VSP, LSP, and TRP had stronger docking interactions than APG. Molecular docking in the ACE active pocket showed that TRP, the most potent ACE inhibitory peptide among the VAP derivatives, had a larger number of interactions with ACE residues in comparison with APG and that the TRP molecule appeared to spread widely in the ACE pocket, while the APG molecule appeared to spread closely. Differences in molecular spread may be a reason why TRP exhibits more potent ACE inhibitory activity than APG. The results suggest that the number and strength of interactions between the peptide and ACE are important for the ACE- inhibitory potency of the peptide.

Unusual clinical manifestations and predominant stopgain ATM gene variants in a single centre cohort of ataxia telangiectasia from North India.

Germline ATM gene variations result in phenotypic heterogeneity characterized by a variable degree of disease severity. We retrospectively collected clinical, genetic, and immunological data of 26 cases with A-T. Clinical manifestations included oculocutaneous telangiectasia (100%), ataxia (100%), fever, loose stools or infection (67%), cerebellar atrophy (50%), nystagmus (8%), dysarthria (15.38%), and visual impairment (8%). Genetic analysis confirmed ATM gene variations in 16 unrelated cases. The most common type of variation was stopgain variants (56%). Immunoglobulin profile indicated reduced IgA, IgG, and IgM in 94%, 50%, and 20% cases, respectively. T cell lymphopenia was observed in 80% of cases among those investigated. Unusual presentations included an EBV-associated smooth muscle tumour located in the liver in one case and Hyper IgM syndrome-like presentation in two cases. Increased immunosenescence was observed in T-cell subsets (CD4+CD57+ and CD8+CD57+). T-cell receptor excision circles (TRECs) were reduced in 3/8 (37.50%) cases.

Epidemiological investigation of spinal muscular atrophy in Japan.

BACKGROUND: International reporting of epidemiological surveys of spinal muscular atrophy (SMA) in Japan has been limited to Shikoku, despite the epidemiology of the disease in countries worldwide becoming clearer. Treatments of 5q-SMA have been developed, and epidemiological studies are needed. PURPOSE: This study aimed to conduct a nationwide epidemiological survey of SMA in Japan to clarify the actual situation of SMA in Japan. METHOD: Patients with all clinical types of SMA, including neonates and adults, were selected from 1,005 medical facilities in Japan. RESULTS: As of December 2017, the actual number of reported patients with SMA was 658 and the genetic testing rate was 79.5%. The estimated number of patients was 1,478 (95% confidence interval (CI), 1,122-1,834), with a prevalence of 1.17 (95%CI, 0.89-1.45) per 100,000 people and an incidence of 0.51 (95%CI, 0.32-0.71) per 10,000 live births. Incidence rates of 5q-SMA by clinical type were 0.27 (95%CI, 0.17-0.38) and 0.08 (95%CI, 0.04-0.11) per 10,000 live births for type 1 and 2, respectively, in cases with a definitive diagnosis by genetic testing. We found that 363 cases (82.7%) occurred less than 2 years and 88 (20.0%) occurred age of 2 months old or under. CONCLUSION: This study clarifies the prevalence and incidence of SMA in Japan. As infantile onset accounts for most cases of SMA, newborn screening and subsequent treatment are important to save lives.

Design and Synthesis of Novel Peptides to Protect Ferulic Acid against Ultraviolet Radiation Based on Domain Site IIA of Bovine Serum Albumin.

Ferulic acid (FA) is known for its excellent antioxidant properties, which can provide many health benefits. One of its drawbacks is its instability under UVA light, which limits its potency. In this study, the new peptides LW2 (QNKRFYFRKNQ) and CW2 (a cyclic form of LW2) were designed based on bovine serum albumin site IIA conformation. A UVA irradiation experiment was performed to investigate the protective ability of these peptides towards FA against UVA damage. The percentages of FA remaining under UV irradiation due to the protection of CW2 and LW2 were 83% and 76%, respectively. The results showed the importance of the cationic residues and hydrophobic residues included in the peptide sequences. Moreover, the cyclic rigid structure showed greater protective ability as compared to its linear counterpart.

Immunological abnormalities in patients with early-onset ataxia with ocular motor apraxia and hypoalbuminemia.

Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH) is a neurodegenerative disorder caused by mutation in the aprataxin (APTX)-coding gene APTX, which is involved in DNA single-strand break repair (SSBR). The neurological abnormalities associated with EAOH are similar to those observed in patients with ataxia-telangiectasia. However, the immunological abnormalities in patients with EAOH have not been described. In this study, we report that EAOH patients have immunological abnormalities, including lymphopenia; decreased levels of CD4+ T-cells, CD8+ T-cells, and B-cells; hypogammaglobulinemia; low T-cell recombination excision circles and kappa-deleting element recombination circles; and oligoclonality of T-cell receptor ß-chain variable repertoire. These immunological abnormalities vary among the EAOH patients. Additionally, mild radiosensitivity in the lymphocytes obtained from the patients with EAOH was demonstrated. These findings suggested that the immunological abnormalities and mild radiosensitivity evident in patients with EAOH could be probably caused by the DNA repair defects.

Axonal damage and behavioral deficits in rats with repetitive exposure of the brain to laser-induced shock waves: Effects of inter-exposure time.

Much attention has been given to effects of repeated exposure to a shock wave as a possible factor causing severe higher brain dysfunction and post-traumatic stress disorder (PTSD)-like symptoms in patients with mild to moderate blast-induced traumatic brain injury (bTBI). However, it is unclear how the repeated exposure and the inter-exposure time affect the brain. In this study, we topically applied low-impulse (∼54 Pa·s) laser-induced shock waves (LISWs; peak pressure, ∼75.7 MPa) to the rat brain once or twice with the different inter-exposure times (15 min, 1 h, 3 h, 24 h and 7 days) and examined anxiety-related behavior and motor dysfunction in the rats as well as expression of ß-amyloid precursor protein (APP) as an axonal damage marker in the brains of the rats. The averaged APP expression scores for the rat brains doubly-exposed to LISWs with inter-exposure times from 15 min to 24 h were significantly higher than those for rats with a single exposure (P < 0.0001). The rats with double exposure to LISWs showed significantly more frequent anxiety-related behavior (P < 0.05) and poorer motor function (P < 0.01) than those of rats with a single exposure. When the inter-exposure time was extended to 7 days, however, the rats showed no significant differences either in axonal damage score or level of motor dysfunction. The results suggest that the cumulative effects of shock wave-related brain injury can be avoided with an appropriate inter-exposure time. However, clinical bTBI occurs in much more complex environments than those in our model. Further study considering other factors, such as the effects of acceleration, is needed to know the clinically-relevant, necessary inter-exposure time.

Clinical, Immunological, and Molecular Features of Severe Combined Immune Deficiency: A Multi-Institutional Experience From India.

Background: Severe Combined Immune Deficiency (SCID) is an inherited defect in lymphocyte development and function that results in life-threatening opportunistic infections in early infancy. Data on SCID from developing countries are scarce. Objective: To describe clinical and laboratory features of SCID diagnosed at immunology centers across India. Methods: A detailed case proforma in an Excel format was prepared by one of the authors (PV) and was sent to centers in India that care for patients with primary immunodeficiency diseases. We collated clinical, laboratory, and molecular details of patients with clinical profile suggestive of SCID and their outcomes. Twelve (12) centers provided necessary details which were then compiled and analyzed. Diagnosis of SCID/combined immune deficiency (CID) was based on 2018 European Society for Immunodeficiencies working definition for SCID. Results: We obtained data on 277 children; 254 were categorized as SCID and 23 as CID. Male-female ratio was 196:81. Median (inter-quartile range) age of onset of clinical symptoms and diagnosis was 2.5 months (1, 5) and 5 months (3.5, 8), respectively. Molecular diagnosis was obtained in 162 patients - IL2RG (36), RAG1 (26), ADA (19), RAG2 (17), JAK3 (15), DCLRE1C (13), IL7RA (9), PNP (3), RFXAP (3), CIITA (2), RFXANK (2), NHEJ1 (2), CD3E (2), CD3D (2), RFX5 (2), ZAP70 (2), STK4 (1), CORO1A (1), STIM1 (1), PRKDC (1), AK2 (1), DOCK2 (1), and SP100 (1). Only 23 children (8.3%) received hematopoietic stem cell transplantation (HSCT). Of these, 11 are doing well post-HSCT. Mortality was recorded in 210 children (75.8%). Conclusion: We document an exponential rise in number of cases diagnosed to have SCID over the last 10 years, probably as a result of increasing awareness and improvement in diagnostic facilities at various centers in India. We suspect that these numbers are just the tip of the iceberg. Majority of patients with SCID in India are probably not being recognized and diagnosed at present. Newborn screening for SCID is the need of the hour. Easy access to pediatric HSCT services would ensure that these patients are offered HSCT at an early age.

A synonymous splice site mutation in IL2RG gene causes late-onset combined immunodeficiency.

X-Linked severe combined immunodeficiency (X-SCID) is a severe form of primary immunodeficiency characterized by absence of T cells and NK cells. X-SCID is caused by a loss-of-function mutation in the IL2RG gene that encodes common gamma chain (γc), which plays an essential role in lymphocyte development. We report the first case of hypomorphic X-SCID caused by a synonymous mutation in the IL2RG gene leading to a splice anomaly, in a family including two patients with diffuse cutaneous warts, recurrent molluscum contagiosum, and mild respiratory infections. The mutation caused aberrant splicing of IL2RG mRNA, subsequently resulted in reduced γc expression. The leaky production of normally spliced IL2RG mRNA produced undamaged protein; thus, T cells and NK cells were generated in the patients. Functional assays of the patients' T cells and NK cells revealed diminished cytokine response in the T cells and absent cytokine response in the NK cells. In addition, the TCR repertoire in these patients was limited. These data suggest that a fine balance between aberrant splicing and leaky production of normally spliced IL2RG mRNA resulted in late-onset combined immunodeficiency in these patients.

Hematopoietic stem cell transplantation for progressive combined immunodeficiency and lymphoproliferation in patients with activated phosphatidylinositol-3-OH kinase δ syndrome type 1.

BACKGROUND: Activated phosphatidylinositol-3-OH kinase δ syndrome type 1 (APDS1) is a recently described primary immunodeficiency syndrome characterized by recurrent respiratory tract infections, lymphoid hyperplasia, and Herpesviridae infections caused by germline gain-of-function mutations of PIK3CD. Hematopoietic stem cell transplantation (HSCT) can be considered to ameliorate progressive immunodeficiency and associated malignancy, but appropriate indications, methods, and outcomes of HSCT for APDS1 remain undefined. OBJECTIVE: Our objective was to analyze the clinical manifestations, laboratory findings, prognosis, and treatment of APDS1 and explore appropriate indications and methods of HSCT. METHODS: We reviewed retrospectively the medical records of cohorts undergoing HSCT at collaborating facilities. RESULTS: Thirty-year overall survival was 86.1%, but event-free survival was 39.6%. Life-threatening events, such as severe infections or lymphoproliferation, were frequent in childhood and adolescence and were common indications for HSCT. Nine patients underwent HSCT with fludarabine-based reduced-intensity conditioning. Seven patients survived after frequent adverse complications and engraftment failure. Most symptoms improved after HSCT. CONCLUSION: Patients with APDS1 showed variable clinical manifestations. Life-threatening progressive combined immunodeficiency and massive lymphoproliferation were common indications for HSCT. Fludarabine-based reduced-intensity conditioning-HSCT ameliorated clinical symptoms, but transplantation-related complications were frequent, including graft failure.

Clinical and Immunological Characterization of ICF Syndrome in Japan.

OBJECTIVE: Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare autosomal recessive primary immunodeficiency. Hypogammaglobulinemia is a major manifestation of ICF syndrome, but immunoglobulin replacement therapy does not seem to be effective for some ICF patients. Therefore, we aimed to reassess the immunological characteristics of this syndrome. METHODS: Eleven Japanese patients with ICF syndrome were enrolled. We performed whole-exome sequencing in four cases and homozygosity mapping using SNP analysis in two. We evaluated their clinical manifestations and immunological status. RESULTS: We newly diagnosed six ICF patients who had tentatively been diagnosed with common variable immunodeficiency. We identified two novel mutations in the DNMT3B gene and one novel mutation in the ZBTB24 gene. All patients showed low serum IgG and/or IgG2 levels and were treated by periodic immunoglobulin replacement therapy. Three of the six patients showed worse results of the mitogen-induced lymphocyte proliferation test. Analyses of lymphocyte subpopulations revealed that CD19+CD27+ memory B cells were low in seven of nine patients, CD3+ T cells were low in three patients, CD4/8 ratio was inverted in five patients, CD31+ recent thymic emigrant cells were low in two patients, and CD19+ B cells were low in four patients compared with those in the normal controls. ICF2 patients showed lower proportions of CD19+ B cells and CD16+56+ NK cells and significantly higher proportions of CD3+ T cells than ICF1 patients. T cell receptor excision circles were undetectable in two patients. Despite being treated by immunoglobulin replacement therapy, three patients died of influenza virus, fatal viral infection with persistent Epstein-Barr virus infection, or JC virus infection. One of three dead patients showed normal intelligence with mild facial anomaly. Two patients presented with autoimmune or inflammatory manifestations. Infectious episodes decreased in three patients who were started on trimethoprim-sulfamethoxazole and/or antifungal drugs in addition to immunoglobulin replacement therapy. These patients might have suffered from T cell immunodeficiency. CONCLUSION: These results indicate that patients with ICF syndrome have a phenotype of combined immunodeficiency. Thus, to achieve a better prognosis, these patients should be treated as having combined immunodeficiency in addition to receiving immunoglobulin replacement therapy.

Dual emissive bispyrene peptide probes for highly sensitive measurements of trypsin activity and evaluation of trypsin inhibitors.

Peptide substrates were double labeled with pyrenes to prepare fluorescent probes for highly sensitive detection of protease activity and evaluation of protease inhibitors using pyrene monomer/excimer signals. Two proximate pyrene moieties formed excited state dimers in the probes, and these pyrene excimer formations were dissociated by tryptic digestion. The specificity constant of the optimum bispyrene peptide probe was 2.7 times higher than that of the conventional peptide-4-methylcoumarin amide. Moreover, our probe had high sensitivity with an estimated detection limit for trypsin of 4.11 pM. The half maximal inhibitory concentration and dissociation constant of the Bowman-Birk inhibitor were successfully estimated.

Efficient near infrared fluorescence detection of elastase enzyme using peptide-bound unsymmetrical squaraine dye.

Extended wavelength analyte-responsive fluorescent probes are highly desired for the imaging applications owing to their deep tissue penetration, and minimum interference from autofluorescence by biomolecules. Near infra-red (NIR) sensitive and self-quenching fluorescent probe based on the dye-peptide conjugate (SQ 1 PC) was designed and synthesized by facile and efficient one-pot synthetic route for the detection of Elastase activity. In the phosphate buffer solution, there was an efficient quenching of fluorescence of SQ 1 PC (86%) assisted by pronounced dye-dye interaction due to H-aggregate formation. Efficient and fast recovery of this quenched fluorescence of SQ 1 PC (> 50% in 30s) was observed on hydrolysis of this peptide-dye conjugate by elastase enzyme. Presently designed NIR sensitive self-quenching substrate offers the potential application for the detection of diseases related to proteases by efficient and fast detection of their activities.

Novel fluorescent substrates for detection of trypsin activity and inhibitor screening by self-quenching.

A group of self-quenching-based substrates with two fluorescent peptides for detection of trypsin activity was designed and synthesized. The substrates could be easily synthesized using simple solid-phase peptide synthesis techniques. Two fluorescent peptide substrates for trypsin were conjugated to the amino groups of lysine as a branched unit. The fluorescence of these substrates was self-quenched owing to the highly assembled fluorophores on the substrates. The release of these concentrated fluorophores by proteases allows for fluorescence recovery. Self-quenching reduced the fluorescence of the substrates by 64.1%, and the fluorescence intensity was recovered by the release of the fluorophores from the substrate peptides via tryptic cleavage. The kinetic assay revealed that the kcat/Km values of the substrates were almost comparable to those of the standard fluorescent probe, peptide-MCA. The detection limit for trypsin was 111pM, and the calculation of IC50 and Ki values for the Bowman-Birk inhibitor was achieved using these substrates. These easily synthesizable self-quenching-based substrates have the potential to be useful for the detection of other disease-related protease activities.

Phosphatase and tensin homolog (PTEN) mutation can cause activated phosphatidylinositol 3-kinase δ syndrome-like immunodeficiency.

BACKGROUND: Activated phosphatidylinositol 3-kinase δ syndrome (APDS) is a recently discovered primary immunodeficiency disease (PID). Excess phosphatidylinositol 3-kinase (PI3K) activity linked to mutations in 2 PI3K genes, PIK3CD and PIK3R1, causes APDS through hyperphosphorylation of AKT, mammalian target of rapamycin (mTOR), and S6. OBJECTIVE: This study aimed to identify novel genes responsible for APDS. METHODS: Whole-exome sequencing was performed in Japanese patients with PIDs. Immunophenotype was assessed through flow cytometry. Hyperphosphorylation of AKT, mTOR, and S6 in lymphocytes was examined through immunoblotting, flow cytometry, and multiplex assays. RESULTS: We identified heterozygous mutations of phosphatase and tensin homolog (PTEN) in patients with PIDs. Immunoblotting and quantitative PCR analyses indicated that PTEN expression was decreased in these patients. Patients with PTEN mutations and those with PIK3CD mutations, including a novel E525A mutation, were further analyzed. The clinical symptoms and immunologic defects of patients with PTEN mutations, including lymphocytic AKT, mTOR, and S6 hyperphosphorylation, resemble those of patients with APDS. Because PTEN is known to suppress the PI3K pathway, it is likely that defective PTEN results in activation of the PI3K pathway. CONCLUSION: PTEN loss-of-function mutations can cause APDS-like immunodeficiency because of aberrant PI3K pathway activation in lymphocytes.