Pre-pubertal exposure of cytarabine-induced testicular atrophy,impaired spermatogenesis and germ cell DNA damage in SD rats

Context: Cytarabine (Ara-C) is an effective chemotherapeutic drug for the treatment of acute leukaemias. It inhibits the DNA synthesis and repair, thereby causes cytotoxicity in the proliferating cells.

Objective: This study was aimed to investigate the effects of pre-pubertal exposure of Ara-C on testesticular development in juvenile SD rats and their function at puberty.

Materials and methods: Ara-C was injected at the doses of 50, 100 and 200?mg/kg/day from postnatal day (PND) 29–42 (14 days) by intraperitoneal (i.p.) route. Half of the animals were sacrificed on PND 43 and remaining on PND 70. End points of the evaluation included gross pathological examination, histomorphometric analysis, sperm count and sperm head morphology, cell proliferation and DNA damage as well as apoptosis analysis.

Results: Ara-C treatment significantly decreased food and water intake, weight gain, testes and epididymis weight and increased histological alterations in the seminiferous tubule. Furthermore, Ara-C treatment significantly decreased the PCNA-positive cells and sperm count in a dose-dependent manner. Ara-C treatment also increased the DNA damage and apoptosis in testes and sperm as evident from the comet and TUNEL assays results.

Discussion: The present study results clearly indicated that Ara-C treatment impaired spermatogenesis and adversely affects the testicular development and its function in rats by reducing the germ cell proliferation and the inducing DNA damage and apoptosis.     

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry–based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.The kidney filter consists of three layers: fenestrated endothelial cells, the glomerular basement membrane, and podocytes.¹ Damage to any of these compartments becomes clinically evident as proteinuria and the development of kidney disease.² Of particular importance for the regulation of podocyte biology through signaling is the slit diaphragm, a specialized intercellular junction that bridges the 40-nm gap in between foot processes of neighboring podocytes. It also serves as a signaling platform regulating podocyte function. Mutations in genes encoding for components of the slit diaphragm, such as nephrin,³ podocin,⁴ CD2AP,⁵ and TRPC6,^6,7 are important causes of genetic forms of proteinuria. Alteration of these proteins results in defective signaling causing podocyte dysfunction, progressive glomerulosclerosis, and kidney failure. The slit diaphragm protein complex is a lipid-multiprotein supercomplex.⁸ Of central importance to the integrity and function of the protein complex is the prohibitin homology (PHB) domain protein podocin,⁹ which forms multimeric complexes and is required to control signal transduction through associated transmembrane proteins.^10,11Signaling processes governing podocyte function, integrity, and survival largely depend on signaling processes involving phosphorylation.^12,13 Comprehensive analyses of the signaling events in podocytes in vivo have been hampered by the fact that interference with these signaling cascades by genetic deletion often results in massively disrupted and dysfunctional podocytes. One of the primary aims of this study was to use phosphoproteomics to analyze thousands of phosphorylation sites in native murine glomeruli within single samples. Within this study, we show that this approach allows the introduction of new concepts into signaling processes at the kidney filtration barrier.     

Evaluation of aqueous extract of Murraya koenigii in unilateral renal ischemia reperfusion injury in rats

Aim:

The aqueous extract of leaves of Murraya koenigii was studied for its renoprotective potential against unilateral renal ischemia reperfusion (RIR) injury in male Wistar rats.

Materials and Methods:

Healthy adult male Wistar rats were divided into five groups (n = 8) and were treated with 200 mg/kg., p.o. of aqueous extract of M. koenigii (AEMK) for 30 days to assess both preventive and curative effects of AEMK. Except Group I, RIR was induced to all the groups by clamping the left renal artery using artery clamp for 1 h followed by reperfusion by removing the clamp. Groups II and III underwent RIR at 30^th day whereas RIR was induced in Groups IV and V at 1^st day of treatment schedule. Biochemical parameters (serum creatinine, blood urea nitrogen, serum total protein and serum Na⁺), urinary parameters (urine output, urinary creatinine, urinary urea, urinary total protein, urinary Na⁺), in vivo anti-oxidants, renal myeloperoxidase (MPO) activity and histopathology of kidneys were monitored. Statistical significance was set at P < 0.05.

Results:

Rats were treated with AEMK significantly (P < 0.05) restored the serum and urinary parameters with significant (P < 0.05) improvement in endogenous anti-oxidants such as superoxide dismutase, catalase and reduced glutathione and decreased levels of malondialdehyde and renal MPO when compared with the control groups. Histopathological examination also supported the biochemical and urinary tests.

Conclusions:

Aqueous extract of M. koenigii possesses both preventive and curative effects against RIR injury.KEY WORDS: Aqueous extract, ischemia reperfusion injury, kidney, Murraya koenigii     

Malnutrition after pancreatic enzyme replacement therapy in chronic pancreatitis: Risk factors in real world practice

BackgroundRCTs that have shown improvement in coefficient of fat absorption with pancreatic enzyme replacement therapy (PERT) have seldom evaluated the impact on overall nutritional status.ObjectiveIn this study we evaluated factors responsible for persistence of malnutrition after PERT.MethodsIn this cross-sectional observational study, patients were enrolled based on predefined enrolment criteria. Patients were divided into those taking PERT regularly (Group A), irregularly (Group B) and not taking (Group C) for at least 3 months. Comprehensive evaluation of anthropometric measurements, nutritional assessment and dietary intake was performed. Malnutrition was measured using the Subjective Global Assessment (SGA) tool. Relationship between PERT status, dietary intake and nutritional status were evaluated using standard statistical methods. Logistic regression was performed to identify factors associated with persistence of malnutrition after PERT.Results377 patients with CP and 50 controls were included. 95 (25.2%) patients with CP were in Group A, 106 (28.1%) in Group B and 176 (46.7%) in Group C. 130 (34.5%) patients were malnourished, of which 76 (58.5%) were continuing PERT. There were no differences in clinical and biochemical nutritional markers between Groups A, B, and C. Calorie deficit and daily intake of calorie, protein, carbohydrates and fats were not different between those with and without PERT, but was significantly less in those with malnutrition. Logistic regression demonstrated inadequate dietary intake as independent risk factor for persistence of malnutrition.ConclusionEven though PERT is effective in PEI, comprehensive nutritional assessment, personalized nutritional counselling and therapy along with PERT is mandatory.     

Challenges in optimizing sample preparation and LC‐MS/MS conditions for the analysis of carglumic acid,an N‐acetyl glutamate derivative in human plasma

This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed‐phase solid‐phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic‐lipophilic balance, and mixed‐mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed‐mode anion exchange (~ 50%) cartridge, compared to other sorbents from 100 μL plasma sample. Complete analytical separation of carglumic acid and carglumic acid‐13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 × 4.6 mm, 5 µm) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) ? 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor → product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00‐6000 ng/mL (r² ≥ 0.9987). Matrix effect was evaluated in terms of IS‐normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra‐ and inter‐batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject. Copyright © 2015 John Wiley & Sons, Ltd.     

Chronic low-level arsenic exposure reduces lung function in male population without skin lesions

Objectives

The respiratory effects of chronic low-level arsenic exposure from groundwater have been investigated in West Bengal, India.

Methods

The participants (834 non-smoking adult males) were subdivided in two groups: an arsenic-exposed group (n = 446, mean age 35.3 years) drinking arsenic-contaminated groundwater (11–50 μg/L) and a control group of 388 age-matched men drinking water containing <10 μg/L of arsenic. Arsenic in water samples was measured by atomic absorption spectroscopy. The prevalence of respiratory symptoms was documented by structured, validated questionnaire. Pulmonary function test (PFT) was assessed by portable spirometer.

Results

Compared with control, the arsenic-exposed subjects had higher prevalence of upper and lower respiratory symptoms, dyspnea, asthma, eye irritation and headache. Besides, 20.6 % of arsenic-exposed subjects had lung function deficits (predominantly restrictive and combined types) compared with 13.6 % of control (p < 0.05). A positive association was observed between arsenic concentration in drinking water and the prevalence of respiratory symptoms, while a negative association existed between arsenic level and spirometric parameters.

Conclusions

The findings suggest that even low-level arsenic exposure has deleterious respiratory effects.     

Integrity of medial temporal structures may predict better improvement of spatial neglect with prism adaptation treatment

Prism adaptation treatment (PAT) is a promising rehabilitative method for functional recovery in persons with spatial neglect. Previous research suggests that PAT improves motor-intentional “aiming” deficits that frequently occur with frontal lesions. To test whether presence of frontal lesions predicted better improvement of spatial neglect after PAT, the current study evaluated neglect-specific improvement in functional activities (assessment with the Catherine Bergego Scale) over time in 21 right-brain-damaged stroke survivors with left-sided spatial neglect. The results demonstrated that neglect patients’ functional activities improved after two weeks of PAT and continued improving for four weeks. Such functional improvement did not occur equally in all of the participants: Neglect patients with lesions involving the frontal cortex (n?=?13) experienced significantly better functional improvement than did those without frontal lesions (n?=?8). More importantly, voxel-based lesion-behavior mapping (VLBM) revealed that in comparison to the group of patients without frontal lesions, the frontal-lesioned neglect patients had intact regions in the medial temporal areas, the superior temporal areas, and the inferior longitudinal fasciculus. The medial cortical and subcortical areas in the temporal lobe were especially distinguished in the “frontal lesion” group. The findings suggest that the integrity of medial temporal structures may play an important role in supporting functional improvement after PAT.     

Society of Abdominal Radiology (SAR) and European Society of Urogenital Radiology (ESUR) joint consensus statement for MR imaging of placenta accreta spectrum disorders

This study was conducted in order to establish the joint Society of Abdominal Radiology (SAR) and European Society of Urogenital Radiology (ESUR) guidelines on placenta accreta spectrum (PAS) disorders and propose strategies to standardize image acquisition, interpretation, and reporting for this condition with MRI. The published evidence-based data and the opinion of experts were combined using the RAND–UCLA Appropriateness Method and formed the basis for these consensus guidelines. The responses of the experts to questions regarding the details of patient preparation, MRI protocol, image interpretation, and reporting were collected, analyzed, and classified as “recommended” versus “not recommended” (if at least 80% consensus among experts) or uncertain (if less than 80% consensus among experts). Consensus regarding image acquisition, interpretation, and reporting was determined using the RAND–UCLA Appropriateness Method. The use of a tailored MRI protocol and standardized report was recommended. A standardized imaging protocol and reporting system ensures recognition of the salient features of PAS disorders. These consensus recommendations should be used as a guide for the evaluation of PAS disorders with MRI. • MRI is a powerful adjunct to ultrasound and provides valuable information on the topography and depth of placental invasion. • Consensus statement proposed a common lexicon to allow for uniformity in MRI acquisition, interpretation, and reporting of PAS disorders. • Seven MRI features, namely intraplacental dark T2 bands, uterine/placental bulge, loss of low T2 retroplacental line, myometrial thinning/disruption, bladder wall interruption, focal exophytic placental mass, and abnormal vasculature of the placental bed, reached consensus and are categorized as “recommended” for diagnosing PAS disorders.     

CCR9 signaling in dendritic cells drives the differentiation of Foxp3+ Tregs and suppresses the allergic IgE response in the gut

The chemokine receptor CCR9 and its only known ligand CCL25 play an important role in gut inflammation and autoimmune colitis. The function of CCR9-CCL25 in the migration of immune cells is well characterized. However, its role in the immune cell differentiation is mostly not known. Using dextran sodium sulfate (DSS)-induced gut inflammation model, we showed that CCR9⁺ dendritic cells (DCs) specifically CD11b⁻CD103⁺ DCs were significantly increased in the gut-associated lymphoid tissues (GALT) compared to control mice. These CCR9⁺ DCs express lower MHC II and CD86 molecules and had regulatory surface markers (FasL and latency-associated peptide, LAP) in the GALT. In the presence of CCL25, CCR9⁺ DCs promoted in vitro differentiation of Foxp3⁺ regulatory CD4⁺ T cells (Tregs). CCL25-induced differentiation of Tregs was due to intrinsic signaling in the DCs but not through CD4⁺ T cells, which was driven by the production of thymic stromal lymphopoietin (TSLP) and not IL-10. Furthermore, adoptive transfer of CCR9⁺ DCs in C57BL/6 mice promoted Tregs but reduced the Th17 cells in the GALT, and also suppressed the OVA-specific gut-allergic response. Our results suggest CCR9⁺ DCs have a regulatory function and may provide a new cellular therapeutic strategy to control gut inflammation and allergic immune reaction.     

Protein-activated transformation of silver nanoparticles into blue and red-emitting nanoclusters

Proteins are very effective capping agents to synthesize biocompatible metal nanomaterials in situ. Reduction of metal salts in the presence of a protein generates very different types of nanomaterials (nanoparticles or nanoclusters) at different pH. Can a simple pH jump trigger a transformation between the nanomaterials? This has been realized through the conversion of silver nanoparticles (AgNPs) into highly fluorescent silver nanoclusters (AgNCs) via a pH-induced activation with bovine serum albumin (BSA) capping. The BSA-capped AgNPs, stable at neutral pH, undergo rapid dissolution upon a pH jump to 11.5, followed by the generation of blue-emitting Ag₈NCs under prolonged incubation (∼9 days). The AgNPs can be transformed quickly (within 1 hour) into red-emitting Ag₁₃NCs by adding sodium borohydride during the dissolution period. The BSA-capping exerts both oxidizing and reducing properties in the basic solution; it first oxidizes AgNPs into Ag⁺ and then reduces the Ag⁺ ions into AgNCs.

Protein capping can trigger nanoparticle to nanocluster transformation at elevated pH.

Noble metal nanomaterials, especially silver (Ag) and gold (Au), have witnessed exceptional research exploration in the last couple of decades from both fundamental and application perspectives.¹ These nanomaterials mainly exist in two distinct size regimes with unique optical characteristics. Ultra-small nanoclusters (NCs) (size typically <3 nm) contain only a handful of atoms (few to hundred), while relatively large nanoparticles (NPs) may comprise thousands of atoms. NPs may display strong extinction (absorption or scattering) spectra in the UV-vis region but are generally non-fluorescent.² In contrast, metal nanoclusters (MNCs) exhibit bright emission but not so noteworthy absorption spectra.^3,4 The distinct optical characteristics of the two nanomaterials have been exploited in various applications. For example, metal nanoparticles (MNPs) are extensively used in photothermal therapy⁵ and imaging,⁶ while NCs are more suited in fluorescence imagining⁷ and sensing⁸ applications. A facile transformation between the two nanomaterials could enable us to combine the complementary optical properties in a single system. Moreover, the kinetics of transformation can provide insights on various intermediate processes like dissolution, etching and digestive ripening etc.^9–11Silver nanoparticles (AgNPs) and nanoclusters (AgNCs) are of particular interest, as it not only possess the intriguing physicochemical properties of MNPs and MNCs, but also feature unique properties pertaining to silver.^3,12,13 For example, metallic silver has been well known for its capability to prevent infection since the ancient times, while recent studies revealed that ultrasmall AgNCs exhibit even superior antibacterial properties towards a broad spectrum of bacteria.^13,14 Moreover, due to superior plasmonic properties and bright fluorescence, AgNPs and AgNCs are preferred over other metal nanomaterials.^15,16The fluorescence properties of AgNCs mainly be attributed to the quantum confinement effect or surface ligand effect.¹⁷ The strong fluorescence generally arises from the electronic transition between occupied d band and states above the Fermi level (sp bands) or the electronic transition between highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO).¹⁸ Several reviews have been devoted for the fundamental understanding of the fluorescence origin of AgNCs.^17,19 Recently, it was demonstrated that aggregation-induced emission (AIE) may also contribute to the luminescence pathway of MNCs.^19,20 The origin of AIE from MNCs could be attributed to the restriction of intramolecular vibration and rotation of ligand on the surface of MNCs after aggregation, which facilitates the radiative energy relaxation via inhibiting of non-radiative relaxations.^21,22Protein capping is quite common for obtaining both NPs^23,24 and NCs.^25–29 Serum proteins, bovine serum albumin (BSA) and human serum albumin (HSA) are the most popular among trials with different proteins.^26–29 BSA is a large protein which provides steric stabilization to the MNCs with its various functional group like –OH, –NH₂, –COOH, –SH.^25,30 The disulfide bond of BSA may have strong interaction with the MNCs where sulfur may be covalently bonded to the MNCs core.^24,31 The nanomaterials are synthesized within the protein template at very different pHs. AgNPs are obtained from the reduction of silver salts at neutral pH (6–8),²⁴ whereas the same process at a higher pH (>11) leads to AgNCs.^30,32 The protein capping itself may reduce Ag⁺; AgNCs are formed without any external reducing agent.^25,33 However, an external reducing agent may change the nature and kinetics of the NCs significantly.³⁰Thus, the influence of pH on the protein structure may govern the selective synthesis of AgNPs or AgNCs. BSA can achieve several conformations – N (native), B (basic), A (aged) and U (unfolded) as the pH of the medium gradually changes from neutral to highly alkaline.^34,35 It may be possible that a specific type of nanomaterial is stable within a particular conformation dictated by the pH of the medium. Hence, by simply changing the pH, we may expect a significant modulation of the morphology of the nanomaterial. Herein, we applied this concept to show an effortless transformation from AgNP to AgNC. Although BSA template is exceptionally popular in the preparation of both AgNPs and AgNCs, however, to the best of our knowledge, no report is available on the conversion from AgNP to AgNC within the protein capping.The BSA-capped AgNPs (BSA-AgNPs) were first synthesized at a neutral pH (pH = 6) using sodium borohydride reduction (see ESI^†). The AgNPs show a sharp surface plasmon resonance (SPR) band at 415 nm (Fig. 1a) and have uniform diameters of 12.5 ± 1.5 nm (Fig. 1b). The AgNPs are quite stable at this pH with no apparent change in the SPR band even after 15 days (Fig. S1^†).Open in a separate windowFig. 1(a) UV-vis spectrum and (b) TEM image of BSA-protected AgNPs synthesized at pH 6. The insets show the appearance of the AgNP solution under regular and UV light (left panel), and size distribution histogram (right panel).However, when the BSA-AgNPs were treated with NaOH to elevate the pH to 11.5, we observed a remarkable decrease in the SPR band at 415 nm and a color change from dark to light brown within 2 h of the pH jump (Fig. 2a). The observations indicate the dissolution of AgNPs, which was further confirmed from the TEM images taken quickly (∼10 min) after the NaOH treatment (Fig. S2^†). Heterogeneous distribution of AgNPs was obtained with sizes varying from 2.6 nm to 17 nm, which is in sharp contrast to the uniform AgNPs before the addition NaOH (cf.Fig. 1b). Upon further incubation (6 h), the light brown color gradually faded to light yellow with a further decrease in the SPR band absorbance (Fig. S3^†).Open in a separate windowFig. 2Time evaluation of the (a) UV-visible and (b) emission spectra (λ_ex = 370 nm) of BSA-capped AgNPs after enhancement of the pH from 6 to 11.5 (by addition of NaOH at t = 0). The inset shows the snapshot of the final blue-emitting AgNC solution under normal and UV lights. (c) TEM of the blue-emitting AgNCs along with the HRTEM image and the analyzed size distribution in the inset. (d) MALDI-mass spectra of the BSA protein and BSA-capped blue-emitting AgNCs.Interestingly, the solution also develops distinct fluorescence with a maximum at ∼460 nm after the addition of NaOH (Fig. 2b). The fluorescence intensity gradually grows up upon incubation, and finally, an intense blue fluorescence was developed within ∼9 days. The final NaOH-treated AgNP solution appears to be light yellow under normal light and blue-fluorescent when viewed under a hand-held UV lamp (Fig. 2b, inset). The blue-emitting AgNCs exhibit a single band excitation spectrum with a maximum at 372 nm (Fig. S4^†).TEM image of the optimized NCs (after 9 days incubation at 37 °C) exclusively reveals uniform AgNCs of ∼2.10 ± 0.28 nm diameter without any trace of large NPs (Fig. 2c). The mass of the BSA-capped AgNCs (67 375 Da) was shifted by 845 Da from that of native BSA (66 530 Da) (Fig. 2d). Thus, the new species should correspond to Ag₈ cluster. The characteristics of the blue-AgNCs were quite similar to the human serum albumin (HSA)-protected blue-AgNCs, directly prepared from silver salt.³³ However, the formation time of those AgNCs was significantly less (∼10 h) than the present method (∼9 days).³³ Thus, the initial dissolution process, although quite fast, may have a crucial role in the kinetics of the protein-protected NCs. When we performed a similar pH jump experiment on a citrate-stabilized AgNP,³⁶ the extinction spectrum of the AgNPs showed much less variation compared to the BSA-AgNPs. Instead of a strong decrease, the SRP band showed a red-shift with an extended tail indicating aggregation rather than dissolution of NPs (Fig. S5^†).Furthermore, a red-emitting cluster was generated when an external reducing agent, sodium borohydride (NaBH₄), was added during the dissolution process. NaBH₄ was added after ∼11 min of the NaOH addition when the SPR band of BSA-AgNP was already decreased by half (Fig. 3a). The SPR band (λ_max = 415 nm) of AgNP continues to diminish similarly before and after the addition of NaBH₄ (Fig. S3^†). Thus, NaBH₄ may not have any significant effect on the dissolution process of AgNP. However, it has a strong impact on the modulation of the fluorescence; a new fluorescence band was developed at ∼650 nm within a much shorter duration (1 h) (Fig. 3b). The solution exhibits a bright-red fluorescence under a UV lamp (Fig. 3b, inset) with a quantum yield of 3.5%.Open in a separate windowFig. 3Early time evolution of (a) UV-visible and (b) emission spectra (λ_ex = 370 nm) of the BSA-protected AgNPs upon subsequent treatments with NaOH (pH 11.5) and NaBH₄ at t = 0 and 11 min, respectively. The decrease of the SPR band at 415 nm and a concomitant increase of the fluorescence band at ∼650 nm indicates dissolution of the AgNPs and formation of the red-emitting cluster. The inset shows the visuals of the AgNCs formed after 1 h under normal light and UV light. (c) TEM of the red-emitting AgNCs along with the HRTEM image and the analyzed size distribution in the inset. (d) MALDI-mass spectra of the BSA protein and BSA-capped red-emitting AgNCs.TEM measurements of the red-emitting species show homogeneous distribution of AgNCs with ∼2.25 ± 0.25 nm diameter (Fig. 3c). MALDI-mass experiment further assigned the red-emitting species as Ag₁₃ cluster (Fig. 3d). The excitation spectrum (λ_em = 650 nm) displays two distinct peaks at 370 nm and 470 nm, which match closely to the reported excitation peaks of the Ag_13–15 clusters within BSA/HSA capping (Fig. S4^†).^30,32,33 Moreover, the fluorescence decay of the red-emitting-AgNCs converted from AgNP almost matches with those prepared directly from AgNO₃; both display very similar average lifetimes (0.95 ns vs. 0.89 ns) (Fig. S6 and Table S1^†).³⁷Another important observation is that the red-emitting AgNCs have only transient stability at 37 °C. With further incubation, the absorbance at ∼470 nm (characteristic excitation peak of the red-emitting cluster) reduces and the absorbance at 370 nm (excitation peak of the blue-emitting cluster) increases simultaneously (Fig. 4a). The red-emission at 650 nm also decreases gradually with a concomitant increase of a blue emission band at 465 nm (Fig. 4b). Thus, both absorption and emission measurements clearly indicate transformation of red- to blue-emitting clusters which takes up to ∼15 days for completion. The solution finally becomes light yellow and exhibits a bright blue fluorescence under UV light similar to the blue-emitting cluster obtained earlier from the AgNP in the absence of NaBH₄. Interestingly, other characteristics of the regenerated blue-emitting AgNCs (converted from Ag₁₃NCs) also match quite nicely with the directly prepared blue-AgNCs (converted from AgNPs in the absence of NaBH₄). The size of this blue cluster was 2.04 ± 0.12 nm, which is similar to the previously obtained direct blue-emitting cluster (2.10 ± 0.28) (Fig. 4c). Furthermore, MALDI-mass measurement reveals that both the blue-emitting clusters may have the same composition, Ag₈ (Fig. 4d). In addition to this, the average lifetime (0.53 ns) of the blue-emitting AgNCs synthesized from AgNP agrees well to the average lifetime (0.40 ns) of the blue-emitting AgNCs converted from the red-emitting AgNCs (Fig. S7 and Table S2^†). However, the quantum yield (23%) of blue-emitting AgNCs, converted from red-emitting AgNCs, was higher than the quantum yield (18%) of the blue-emitting AgNCs, converted from AgNPs. Since, the emission characteristics of the blue and the red-emitting clusters nearly matches with earlier report, we expect that silver may be present in the zero oxidation state as determined in those studies.^30,31Open in a separate windowFig. 4Transformation of red-emitting to blue-emitting cluster: (a) UV-visible and (b) emission spectra (λ_ex = 370 nm) showing transformation of the BSA-protected red-emitting Ag₁₃NCs (obtained at 1 h) to blue-emitting AgNC upon prolonged incubation. Red and blue arrows respectively denote the decrease/increase of the red/blue cluster absorbance and emission intensity with time. The inset (b) shows a magnified wavelength region in 580–720 nm of the emission spectra. (c) TEM image of the blue-emitting silver nanocluster while its inset shows HRTEM image with size histogram of corresponding silver nanocluster. (d) MALDI-mass spectra of BSA and BSA-containing blue-emitting silver nanocluster synthesized from Ag₁₃NCs.Moreover, the atomic composition of the NCs can be also be estimated from the Jellium model using the equation^38,39E_em = E_Fermi/N^0.33where E_Fermi is the Fermi energy of the metal (Ag), E_em is the emission energy of the MNCs and N is the number of atoms constituting a MNC. Using the model equation, the number of silver atoms for the blue-emitting AgNCs can be predicted as 8.45 (∼8) Ag atoms which is a good agreement with our MALDI data (8 Ag atoms). However, the theoretical calculation estimated as N ∼ 24 for the red-emitting AgNCs, which is not in agreement with the MALDI data (13 Ag atoms). This is because of the well-known deviation of the Jellium model for higher number of Ag atoms in AgNCs because of increase in the electronic screening effects and the harmonic distortion in the potential energy well.¹⁹Although the red-emitting cluster is not very stable at the experimental condition (pH 11.5, 37 °C), it may be easily stabilized by lowering the temperature or pH. The fluorescence intensity of the red-emitting and blue-emitting cluster kept at 4 °C, was almost preserved for more than 15 days (Fig. S8^†). On the other hand, lowering the pH to 6, also inhibits the red to blue-cluster transformation (Fig. S9^†). The observations indicate that the red-blue transformation has a moderate activation barrier and the conversion may be governed by the change in the structure of the protein in the alkaline condition. Acidification of the solution can stop the transformation of the protein conformation and inhibits the process.From these observations, we may conclude that the conversion from NPs to NCs occurs in two steps. First, a rapid dissolution of AgNP occurs in the alkaline medium. The kinetics of the dissolution process can be monitored through a time-dependent decrease of the SPR band and the time constant was found to be ∼13 min (Fig. S10^†). Dissolution of AgNPs is an important issue and assumed to be the leading cause of toxicity of AgNPs in biological mediums.⁴⁰ The dissolution is commonly favored at a low pH but drastically inhibited at high pH.⁴¹ The swift dissolution of the BSA-protected AgNP observed here at a high pH (11.5) is unprecedented. Thus, the BSA capping may have an active role in the dissolution process. We comprehend that the oxidation power of protein may be activated in the basic medium.Organothiols (R-SH) are known to promote dissolution of AgNPs; R-SH progressively reacts with Ag atoms to form RS-Ag complex.⁴² Since cysteine is also an organothiol, it is expected to play an essential role in the dissolution of AgNPs. Gondikas et al. showed that excess cysteine could favor the dissolution process of AgNPs, whereas another amino acid, serine (S–H bond is replaced by O–H bond), has no effect.⁴³ Zang and coworkers showed that only the isolated or reduced cysteine in a protein has a dominant role in the dissolution of NPs.⁴⁴ Although BSA contains as many as 35 cysteine residues; 34 of them are involved in S–S bond formation and only a single cysteine is present in free form (S–H). Hence, the dissolution of AgNPs at neutral pH may be negligible.Most proteins rich in sulfur-containing residues (cysteine and methionine) may degrade in alkaline solution. Florence reported that about 5 of 17 S–S bridges in BSA may be cleaved in the presence of 0.2 M NaOH.⁴⁵ Thus, at higher pH, some disulfide bonds may be cleaved and more cysteine residues may participate in the dissolution of BSA-capped AgNPs.In the second step, Ag⁺ ions generated from the dissolution of AgNPs, can be reduced either by the protein capping itself or by an external reducing agent to form NCs (Scheme 1). The tyrosine residues may be responsible for the reduction of the metal ions to NCs.^25,33 At a pH, higher than the pKa (10.46) of tyrosine, the reduction capability of tyrosine is enhanced by deprotonation of the phenolic group.^25,33,46 Moreover, the addition of a strong reducing agent (e.g., NaBH₄) may lead to a faster reduction, which favors quicker nucleation and growth of Ag atoms forming the bigger NCs (Ag₁₃NCs). However, the large Ag₁₃NCs may not be adequately stabilized by the protein conformation at that condition and hence may transform into the more stable blue-emitting Ag₈NCs.Open in a separate windowScheme 1Schematic representation of the transformation of the BSA-capped AgNPs to blue- and red-emitting AgNCs.The conformation change of the protein capping during the conversion was also supported by the circular dichroism (CD) measurements (Fig. S11^†). The formation of AgNPs results in a negligible change in the protein conformation (Table S3^†). However, the formation of red Ag₁₃ cluster results in a substantial modification in the BSA conformation. The α helix content reduces from 57% to 49%, whereas coil randomness increases from 17% to 21% without a major change in the β sheet. Interestingly, blue-emitting Ag₈ cluster perturbed the conformation of the BSA to a much larger extent (Table S3^†). As the cysteine disulfide bond has a direct role on maintaining the folded conformation of BSA, its breaking may change the protein conformation. The addition of NaOH induces breaking of S–S bond, which leads to formation of AgNCs with subsequent change in protein secondary structure.In conclusion, we report an unprecedented fast dissolution of AgNPs through activation of the protein (BSA) capping by elevating the pH of the medium to 11.5. At higher pH, the disulfide bonds may be cleaved, and the free cysteine may activate the dissolution process. The protein capping also plays a crucial role in the formation of fluorescent nanocluster after the completion of the dissolution process. Thus, we explored multiple roles of the BSA capping – (1) a stable capping agent at neutral pH to stabilize the AgNPs (2) activates the dissolution process probably via oxidative dissolution of the AgNPs (3) adsorbing the nascent silver ions within its scaffold and (4) finally reducing them to fluorescent nanocluster.