Biological Investigations Research Articles

Site‐Selectively Accelerating the Generation of β‐Linked Residue Isoaspartate in Proteins

AbstractIsoaspartate (isoAsp) is a β‐linked residue in proteins spontaneously generated through Asn deamidation or Asp dehydration and significantly affects protein properties. However, the sluggish and site‐nonselective generation of isoAsp residues in proteins severely impedes in‐depth biological investigations as well as the exploitation of its unique β‐linkage features. Herein, we introduce a method that allows site‐selective and rapid generation of isoAsp residues in proteins. This method leverages the genetic incorporation of a side‐chain‐esterified Asp derivative (BnD), which undergoes facile intramolecular arrangement to form the key intermediate, aspartyl succinimide (Suc); subsequent hydrolysis of Suc gives rise to isoAsp as the major product. On native sites of proteins, including Cu/Zn superoxide dismutase and calmodulin, we demonstrate that BnD‐mediated isoAsp formation is faster than Asn deamidation generally by three orders of magnitude.

Neural responses to light stimulation in the octopus arm.

Octopus are known to be visual animals. Beyond functions of the eyes, recent investigations have documented the importance of extraocular photoreception in behavior. Octopus arms have been shown to respond behaviorally to local light exposure with negative phototaxis. Moreover, light-activated chromatophore expansion (LACE) in octopus arms indicates that skin-based photoreception may contribute to light detection. In this study, we used electrophysiological recordings to investigate the neural activity of the arm's axial nerve cord in response to light on the arm. We tested the hypothesis that light stimulates activity of neurons in the arm's axial nerve cord. We also aimed to determine sensitivities to different wavelengths of light. The results showed that the axial nerve cord was strongly responsive to light stimulation of the arm and that the response travels along the length of the axial nerve cord. Blue light generated the strongest neural activity while red and green light also induced responses. Light induced neural activity was mediated through the aboral arm skin and by the oral-side skin and suckers. These findings reveal the role of the skin in the sensory abilities of octopuses and provide insights into the neural mechanisms underlying their response to light. The study underscores the importance of extraocular photoreception in future investigations of cephalopod sensory and behavioral biology.

Genetically Encoded Fluorescent and Bioluminescent Probes for HDAC8.

Protein-based probes constructed via genetically encoding acetyl lysine (AcK) or its close analogs represent an important way to detect protein lysine deacetylases. Existing reported probes exhibit excellent sensitivity to NAD+-dependent sirtuins but lack responsiveness to Zn2+-dependent histone deacetylases (HDACs). Herein, we reformed the probe design by replacing the genetically encoded AcK with trifluoroacetyl lysine (TfAcK) and generated fluorescent and bioluminescent probes that could respond specifically to HDAC8 recombinantly expressed in E. coli and to endogenous HDACs in mammalian cells. We believe these probes would benefit the biological investigation of HDAC8 and promisingly some other HDACs, as well as the discovery of innovative HDAC inhibitors.

Evolving concepts of low-density lipoprotein: From structure to function.

Low-density lipoprotein (LDL) is a central player in atherogenesis and has long been referred to as 'bad cholesterol.' However, emerging evidence indicates that LDL functions in multifaceted ways beyond cholesterol transport that include roles in inflammation, immunity, and cellular signaling. Understanding LDL's structure, metabolism and function is essential for advancing cardiovascular disease research and therapeutic strategies. This narrative review examines the history, structural properties, metabolism and functions of LDL in cardiovascular health and disease. We analyze key milestones in LDL research, from its early identification to recent advancements in molecular biology and omics-based investigations. Structural and functional insights are explored through imaging, proteomic analyses and lipidomic profiling, providing a deeper understanding of LDL heterogeneity. Low-density lipoprotein metabolism, from biosynthesis to receptor-mediated clearance, plays a crucial role in lipid homeostasis and atherogenesis. Beyond cholesterol transport, LDL contributes to plaque inflammation, modulates adaptive immunity and regulates cellular signaling pathways. Structural studies reveal its heterogeneous composition, which influences its pathogenic potential. Evolving perspectives on LDL redefine its clinical significance, affecting cardiovascular risk assessment and therapeutic interventions. A holistic understanding of LDL biology challenges traditional perspectives and underscores its complexity in cardiovascular health. Future research should focus on further elucidating LDL's structural and functional diversity to refine risk prediction models and therapeutic strategies, ultimately improving cardiovascular outcomes.

Site-Selectively Accelerating the Generation of β-Linked Residue Isoaspartate in Proteins.

Isoaspartate (isoAsp) is a β-linked residue in proteins spontaneously generated via Asn deamidation or Asp dehydration and significantly affects protein properties. However, the sluggish and site-nonselective generation of isoAsp residues in proteins severely impedes the in-depth biological investigations as well as the exploitation of its unique β-linkage features. Herein, we introduced a method that allows site-selective and rapid generation of isoAsp residues in proteins. This method leverages the genetic incorporation of a side-chain esterified Asp derivative (BnD), which undergoes facile intramolecular arrangement to form the key intermediate, aspartyl succinimide (Suc); subsequent hydrolysis of Suc gives rise to isoAsp as the major product. On native sites of proteins including Cu/Zn superoxide dismutase and calmodulin, we demonstrated that BnD-mediated isoAsp formation is faster than Asn deamidation generally by three orders of magnitude.

In vitro and in silico evaluation of synthetic compounds derived from bi-triazoles against asexual and sexual forms of Plasmodium falciparum

BackgroundDespite advances in malaria chemotherapy, the disease continues to claim thousands of lives annually. Addressing this issue requires the discovery of new compounds to counteract resistance threatening the current therapeutic arsenal. In this context, bi-triazoles are substances with diverse biological activities, showing promise as lead compound to fight malaria. Triazoles are heterocyclic structures composed of five members, including three nitrogen atoms and two double bonds. Bi-triazoles, the focus of this study, are derivatives of triazoles consisting of two triazole rings (nitrogen heterocyclic) with isolated nuclei lacking a spacer and two substituents at each end. The goal of the present study was to assess the in vitro and in silico, antimalarial activity of bi-triazole compounds 14c, 14d, 13c, and 13d against asexual and sexual forms of Plasmodium falciparum.MethodsFor in silico predictions, the software OSIRIS, Molinspiration, and ADMETlab were employed. To determine the 50% inhibitory concentration (IC50) on the asexual forms, the W2 clone was used, while the strain NF54 was used to assess inhibition of sexual forms. Cytotoxicity was evaluated using the HepG2 cell line, and haemolysis tests were conducted. Additionally, the selectivity index (SI) of each compound was calculated.ResultsIn silico analyses of physicochemical properties revealed that all compounds have favorable potential for drug development. Pharmacokinetics predictions also provided important, novel insights into this chemical class. Antimalarial activity tests showed that compounds 14d and 13d exhibited promising activity, with IC50 values of 3.1 and 4.4 µM, respectively. Antimalarial activity of compounds 14d and 13d may be related to the presence of methyl acetate in substituent R2 conjugated to the bi-triazole. None of the compounds demonstrated cytotoxic or haemolytic activity, with SI values above 51 for the three most active compounds, highlighting their selectivity. For the sexual forms, compounds 14c and 14d were classified as having a high potential to block malaria transmission.ConclusionOverall, the in vitro and in silico results showed that bi-triazole compounds may guide new biological investigation for malaria, enabling the identification and development of more active and selective antimalarial agents.

Structural and biological investigations of Fe(III) and Co(II) complexes with tryptophan and 2,2'-bipyridine: implications for antibacterial and antifungal applications

Abstract This study focused on the design and stractural characterization of two new transition metal complexes derived from Tryptophan (Trp) and 2,2'-bipyridine (Bip), coordinated with Iron(III) (FeTrpBip) and Cobalt(II) (CoTrpBip) ions. Structural elucidation of these complexes was achieved using a range of advanced analytical techniques. Thermal analysis revealed the stability and decomposition behaviors of the complexes. The data indicated that both FeTrpBip and CoTrpBip exhibit octahedral coordination geometries, with the structural formulas identified as [Fe(Trp)(Bip)(Cl)2] and [Co(Trp)(Bip)(Cl)(H2O)], respectively. To support the experimental data, Density Functional Theory (DFT) calculations had been performed. These calculations confirmed the proposed structures and provided a detailed analysis of quantum chemical parameters, including HOMO–LUMO energies, molecular orbitals, and electronic distributions, which are important for understanding the complexes' reactivity. Further, extensive in vitro biological evaluations assessed the antifungal and antibacterial evaluation of the synthesized metal complexes. The bioassays demonstrated that both FeTrpBip and CoTrpBip displayed significantly enhanced bioactivity compared to the free ligands, indicating a synergistic effect of metal coordination on the biological efficacy of the ligands. Molecular docking studies were subsequently conducted to explore the mechanisms of action of these complexes at the molecular level, specifically targeting the E. coli FabH–CoA (PDB ID: 1HNJ). The FabH receptor, essential for fatty acid biosynthesis, was chosen to evaluate the antimicrobial potential of the complexes. Docking simulations provided valuable insights into binding affinities, interaction energies, and key amino acid residues involved in the binding process. The results from these extensive studies highlight the significant therapeutic potential of FeTrpBip and CoTrpBip complexes, positioning them as promising reagents for further development in medical science. The observed synergistic effects due to metal coordination underscore the potential for these complexes to advance antimicrobial therapies and address challenges associated with resistant strains.

Chemical and biological investigations on modified gemcitabine by nanoliposome structured on cholesterol, pectin, and phosphatidylcholine as an anticancer drug via a drug delivery system.

Chemical and biological investigations on modified gemcitabine by nanoliposome structured on cholesterol, pectin, and phosphatidylcholine as an anticancer drug via a drug delivery system.

Green Synthesis of NiO Nanoparticles using Pongamia pinnata and their Catalytic Utility in the Synthesis of N-Fmoc/Cbz-protected Amino Acid Derived Sulfides and their Biological Investigations

Introduction:: Synthesis of NiO nanoparticles using environmentally friendly Pongamia pinnata seeds as a source of fuel was demonstrated using a solution combustion approach. Method:: The protocol for the synthesis of NiO NPs is simple and efficient. NiO NPs were utilized as the catalyst for the synthesis of N-protected aminoalkyl sulfides from N-protected alkyl thiols and bromo esters of amino acids. Results:: The NiO NPs were characterized using XRD, SEM, and EDX techniques. N-protected aminoalkyl sulfides were characterized by HRMS, 1H, and 13C NMR techniques and were evaluated for their in vitro antifungal activities, against A. Niger using Fluconazole as a standard. Conclusion:: The current study presents an effective approach for synthesizing a new class of sulfides from N-protected aminoalkyl thiols and bromomethyl esters in the presence of nano NiO as a catalyst.

Quantification of high molecular weight organic carbon concentrations with LC-OCD and PHMOC for biological stability investigation of drinking water produced from surface water.

Quantification of high molecular weight organic carbon concentrations with LC-OCD and PHMOC for biological stability investigation of drinking water produced from surface water.

Physalis peruviana heteropolysaccharide-conjugated selenium nanoparticles: Preparation, characterization, and promising applications in cancer therapy.

Physalis peruviana heteropolysaccharide-conjugated selenium nanoparticles: Preparation, characterization, and promising applications in cancer therapy.

Cancer-Targeting Peptides Functionalized With Polyarginine Enables GRP78-Dependent Cell Uptake and siRNA Delivery Within the DU145 Prostate Cancer Cells.

This study investigated a peptide-based GRP78-targeting strategy for short-interfering (si) RNA delivery in cancer cells. Synthetic fluorescein-labeled amphiphilic peptides composed of the hydrophobic cell surface (cs) GRP78-targeting and hydrophilic, polycationic arginine-rich cell penetrating peptides demonstrated GRP78-dependent cell uptake in the DU145 prostate cancer cells, and to a lesser extent in the non-cancerous human lung fibroblast WI-38 cell line. Mechanistic studies revealed energy-dependent GRP78 receptor-mediated endocytosis of the GRP78-targeting peptide with polyarginine (W1-R9). The cytosolic accumulation of this peptide underscored its potential utility in siRNA delivery. Peptide:siRNA complexes formed stably condensed nanoparticles, with calcium functioning as an ionic stabilizer and additive promoting endosomal siRNA escape for RNA interference (RNAi) activity. Preliminary peptide-based siRNA transfections in the DU145 cells demonstrated that GRP78 knockdown led to an interplay in between pro-survival and cell death outcomes under ER stress induction. Thus, the GRP78-targeting polyarginine peptides enables efficient cell uptake for specific siRNA delivery in the DU145 cells. This class of bio-active synthetic peptides is important for the investigation of cancer biology, leading to the innovation of cancer-targeted gene delivery and therapy approaches.

Live imaging of the extracellular matrix with a glycan-binding fluorophore.

All multicellular systems produce and dynamically regulate extracellular matrices (ECMs) that play essential roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small-molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, non-perturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

Rhabdomyosarcoma Harboring NRAS or HRAS Mutation Arising in Giant Congenital Melanocytic Nevus: Report of 2 Cases.

Nonmelanoma malignancies associated with congenital melanocytic nevi (CMN) are extremely rare, with only 12 reported cases of rhabdomyosarcoma (RMS) to date. We present 2 additional cases of RMS arising in giant CMN, with immunohistochemical and molecular biologic investigations. The first case was a 32-year-old woman with a personal history of melanoma in giant CMN who, after successful treatment and long remission, presented with a new 1-cm nodule within the CMN. Microscopically, the atypical areas exhibited a round cell/alveolar morphology with immunoreactivity for desmin and myogenin, and lacked PAX3/7::FOXO1 fusions typical for alveolar RMS on a reverse transcription polymerase chain reaction analysis. An identical NRAS p.Q61R mutation with comparable variant allele frequency (32% and 44%) was identified in both the nevus and the RMS tissue by next-generation sequencing. The second patient was a 5-year-old girl with a rapidly growing, bleeding, ulcerated 3 × 4 cm interscapular mass within a giant CMN that histologically seemed as a proliferation of pleomorphic spindle, polygonal and epithelioid cells with marked pleomorphism immunoreactive for myogenin, muscle-specific actin, and smooth muscle actin. Next-generation sequencing yielded an HRAS p.Q61R mutation with limited variant allele frequency (7%) in the RMS component, while ATRX p.Q2193* variant was detected in the nevus. Our study is apparently the first report of NRAS and HRAS mutations in tumors with RMS phenotype arisen in CMN.

Seseli foliosum (Somm. et Levier) Manden.-A Comprehensive Phytochemical and Biological Evaluation.

The genus Seseli L. (Apiaceae family) is widespread across Europe and Asia, with ten species identified in Georgia. Among these, Seseli foliosum (Somm. et Levier) Manden., is notable for its unique pharmacological properties. To our knowledge, comprehensive phytochemical and biological investigations have not yet been conducted. The primary aim of this research is to explore the chemical and biological properties of S. foliosum, thereby enhancing its potential applications in medicine and related fields. Different chromatographic techniques were utilized to isolate individual compounds and to identify the chemical composition of S. foliosum MeOH and Et2O extracts from seeds and roots. A battery of biological assays (antimicrobial, antioxidant, enzymatic, anxiolytic, and cytotoxic) were employed to assess the pharmacological properties of the extracts. The results from gas chromatography with mass spectrometry (GC/MS) revealed that both MeOH and Et2O extracts contain a diverse array of compounds, including monoterpenoids, sesquiterpenoids, and phenolic compounds. Furanocoumarin edultin was isolated from the MeOH extract by liquid-liquid separation (LLS). The MeOH extracts exhibited important antioxidant, enzyme inhibitory, and antimicrobial activities with notable efficacy against Staphylococcus aureus (MIC 125 µg/mL) and Candida glabrata (MIC 62.5 µg/mL). Underground Et2O extracts showed advanced cytotoxic activity, particularly against hypopharyngeal carcinoma cells (CC50 22.33 µg/mL and 27.16 µg/mL, respectively). The study provides a wide-range analysis of the phytochemical composition and biological activities of S. foliosum, highlighting its potential as a source of bioactive compounds. These findings contribute to the understanding of the therapeutic potential of S. foliosum and lay the groundwork for further pharmacological and clinical research.

Pericardial Involvement in Hereditary Hemorrhagic Telangiectasia

Hereditary haemorrhagic telangiectasia is a rare disease characterized by cutaneo-mucous and visceral arteriovenous malformations. Cardiac involvement is uncommon and was presented primarily by hyper-output heart failure. Hemorrhagic pericardial effusion, although is extremely rare, can occur during HHT. We report the case of a 48-years-old woman which was hospitalized in 2015 in internal medicine department, Sfax, Tunisia in 2015 for anicteric cholestasis. She noticed a personnel and familial history of recurrent epistaxis. Biologic findings revealed anemia and moderate cholestasis. Viral investigations and immunologic tests were negative. Abdominal tomography showed multiple arterio-venous shunts of the liver. Liver involvement due to Rendu Osler Weber disease was retained. She was treated by ferrous iron, but she was lost to follow up. She was presented in February 2021, with severe anemia (5 g/dL). Physical examination revealed signs of global heart failure. Biological investigations found anemia, inflammatory biological syndrome, cytolysis and cholestasis. Heart ultrasound revealed an abundant pericardial effusion. Only 500cc of hemorrhagic fluid could be aspirated before the needle became blocked. Unfortunately, one week after, re-accumulation of pericardial fluid and worsening occurred. She underwent a partial surgical pericardial excision with pleuropericardial opening. Analysis of the fluid ruled out any infectious cause of this effusion. Histological examination confirmed the vascular dysplasia with signs of hemorrhage and inflammation. The patient was discharged 1 month after surgery with no other bleeding episodes. For her anemia, she received a transfusion of red blood cells. Then, the patient was treated by iron treatment.

Assessment of Spinster homologue 2 (Spns2)-dependent transport of sphingosine-1-phosphate as a therapeutic target.

Sphingosine-1-phosphate (S1P) receptor modulator (SRM) drugs suppress immune system function by disrupting lymphocyte trafficking, but SRMs are broadly immunosuppressive with on-target liabilities. Another strategy to modulate the immune system is to block S1P transport. This study tests the hypothesis that blockers of S1P transport (STBs) mediated by Spinster homologue 2 (Spns2) approximate the efficacy of SRMs without their adverse events. We have discovered and optimized STBs to enable investigations of S1P biology and to determine whether S1P transport is a valid drug target. The STB SLF80821178 was administered to rodents to assess its efficacy in a multiple sclerosis model and to test for toxicities associated with SRMs or Spns2-deficient mice. Further, potential biomarkers of STBs, absolute lymphocyte counts (ALCs) in blood and S1P concentrations in plasma and lymph, were measured. SLF80821178 resembles SRMs in that it is efficacious in a standard multiple sclerosis model but does not evoke bradycardia or lung leakage, common to the SRM drug class. Also, chronic SLF80821178 administration does not affect auditory responses in adult mice despite the neurosensorial hearing defect observed in Spns2-null mice. While both SRM and STB administration decrease ALCs, the maximal effect is less with an STB (45% vs. 90%). STBs have minimal effects on S1P concentration in plasma or thoracic duct lymph. We found nothing to invalidate Spns2-dependent S1P transport as a drug target. Indeed, STBs could be superior to SRMs as a therapy to modulate immune system function.

Mitochondrial Ribosome Regulation Drives Spermatogenesis and Male Fertility.

Mitochondrial Ribosome Regulation Drives Spermatogenesis and Male Fertility.

Impact of DTPA and 3,4,3-LI(1,2-HOPO) on EuIII interactions with renal cells in vitro.

This study represents a first comprehensive investigation on how the decorporation agents CaNa3-DTPA (DTPA) and 3,4,3-LI(1,2-HOPO) (LIHOPO) affect EuIII interactions with human and rat kidney cells in vitro. Cell biological investigations were complemented with physicochemical measurements to correlate cytotoxic impairments with intracellular metal uptake and EuIII speciation. Upon exposure to sole DTPA or LIHOPO, cell viability and morphology are affected in a time- and concentration-dependent manner. For both decorporation agents, detailed EC50 values for renal cells in vitro are reported. Simultaneous application of EuIII+DTPA in the medium leads to formation of the soluble and largely cell impermeable EuDTPA2- complex. At ligand excess, this significantly reduces intracellular EuIII uptake. However, EuDTPA2- was spectroscopically detected also inside cells indicating that small fractions of this complex are able to pass the plasma membrane. When EuIII+LIHOPO is applied to the medium, the soluble EuLIHOPO- complex is formed. In contrast to DTPA, this drastically enhances intracellular EuIII uptake even at ligand deficit demonstrating that EuLIHOPO- is highly cell permeable. Concomitantly, this complex was spectroscopically detected inside cells confirming its plasma membrane passage and intracellular stability. Nevertheless, due to stable EuIII binding, the cell viability is not influenced by the increased intracellular EuIII content. In fact, the applied ligand concentration is much more critical in this regard, emphasizing the need for cytotoxic investigations. Our results improve the knowledge of the cellular interactions of lanthanides ± decorporation agents and demonstrate the combination of in vitro cell culture and spectroscopy being a sophisticated toolbox for this.

Investigation of Water Quality and Suitability for Household and Drinking Purposes in the Water Distribution System of Anah City

Abstract Clean drinking water is a vital cornerstone for the life of an urban city, supporting the health of its residents and promoting economic development. Sustainable water supplies are essential to ensure the continuity of daily life and provide a safe and sustainable environment for the city’s inhabitants therefore, This study comprised an analysis of the water quality and appropriateness for everyday use in Anah’s water distribution network, one of the cities in the Anbar Governorate, situated more than 320 kilometers west of Baghdad. The samples were collected from 12 different sites, including the river’s source of water before treatment, the purification plant’s interior after treatment, and ten samples representing various parts of the city. Throughout the distribution network, the water’s transit was monitored for water quality. Temperature, turbidity, and electrical conductivity were among the physical testing, while tests for chemicals included (pH number, total dissolved salts, salinity, alkalinity, temporary hardness, and concentrations of chloride, sodium, potassium and sulfate ions). Estimates of the trace element concentrations in water samples included measurements of the element (copper, nickel, lead, zinc and cadmium). In addition to a plate count, the biological investigations included coli and fecal coliform bacteria. The results of the physical, chemical, and biological tests indicated that the water in the distribution network is suitable for drinking and household uses and that it is within the acceptable ranges of the standard specifications of the World Health Organization and the standard specifications of the Iraqi standardization and quality control device.