This study explores the molecular structuring of salmon gelatin (SG) with controlled molecular weight produced from salmon skin, and its relationship with its thermal and rheological properties. SG was produced under different pH conditions to produce samples with well-defined high (SGH), medium (SGM), and low (SGL) molecular weight. These samples were characterized in terms of their molecular weight , molecular weight distribution (electrophoresis), amino acid profile, and Raman spectroscopy. These results were correlated with thermal (gelation energy) and rheological properties. SGH presented the higher MW (173 kDa) whereas SGL showed shorter gelatin polymer chains (MW < 65 kDa). Raman spectra and gelation energy suggest that amount of helical structures in gelatin is dependent on the molecular weight, which was well reflected by the higher viscosity and G′ values for SGH. Interestingly, for all the molecular weight and molecular configuration tested, SG behaved as a strong gel (tan δ < 1), despite its low viscosity and low gelation temperature (3–10 °C). Hence, the molecular structuring of SG reflected directly on the thermal and viscosity properties, but not in terms of the viscoelastic strength of gelatin produced. These results give new insights about the relationship among structural features and macromolecular properties , which is relevant to design a low viscosity biomaterial with tailored properties for specific applications. Many tons of fish by-products are generated by the fishing industry globally each year, including bones, guts, heads, tails, scales, and substantial quantities of skin. Fish skin is known to be a good source of collagen and gelatin ,2,3. GelGelatin from fish adapted to cold water, such as those obtained from salmon skin and other species, shows lower gelling temperatures (<15 °C) compared to mammal and warm water fish gelatins . Indeed,Depending on the application where the SG could be used, its properties can be advantageous allowing the development of different strategies to design salmon gelatin-based biomaterials with tailored and engineered functional and technological properties. Indeed, low viscosity gelatins have been successfully used in applications that require small-scale constructs with well-defined geometries and tuned structures such as electrospinning , microflDespite these recent advances in research on SG, limited information is available about its physical properties and functionality when the polymer features well-defined molecular weights produced by various processing conditions. Only recently Diaz-Calderón  reportedThe purpose of this study is therefore to further explore the molecular structuring of salmon gelatin with controlled molecular weight, with the aim to improve the understanding of its effects on macroscopic features such as thermal and rheological properties of this low viscosity underutilized biomaterial. We believe that this information is necessary for a better understanding of the structure–function relationship for the rational design of SG bases materials with tunable properties at the macro scale required for applications with increasing level of complexity.Salmo salar). Salmon skins were provided by a local salmon processing company and stored at −40 °C until further use. Prior the extraction process, the skins were manually cleaned using a sharp knife in order to eliminate all residues of muscle and scales, and then were cut into squared pieces (3 cm × 3 cm). The extraction protocol followed the methodology proposed by Hou and Regenstein , which can be correlated with the MW through a mathematical model described by the Mark-Houwink-Kuhn-Sakurada (MHKS) equation . The determination of MW of the produced salmon gelatin was carried out using the values of a and K reported by Quero et al. [The gelatin samples were diluted in a 0.1 M NaCl solution, at concentrations ranging from 0 to 6 g Le et al.  and Quere et al. . Viscomeo et al. .Complementary to this technique, the molecular weight (MW) distribution of the SG polymer chains was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All the steps for the SDS-PAGE were performed as described before by Díaz-Calderón et al.  using 4–The concentration of the amino acids present in SG samples was determined by reverse-phase high-performance liquid chromatography (RP-HPLC) following the methodology described by Rebane and Herodes  with theThe isoelectric point (pI) of the SG suspensions (0.015% w/v) was determined by assessing changes in the zeta potential at various pH values using a Zetasizer Nano-Z instrument . The pH was automatically adjusted from pH 3 to pH 12 by an automatic titrator attached to the instrument . Two independent runs with triplicate measurements were performed (n = 6).−1. The laser was focused on the sample’s surface using an optical microscope (Olympus BX41) using a ×100 long-working distance objective . Each spectrum was acquired within the wavenumber range of 200–3500 cm−1 using an exposure time of 20 s and three accumulations. All spectra were normalized and corrected using the instrument software . Five replicates were used for each measurement.For this analysis, SG suspensions at a concentration of 7% w/v were prepared at 60 °C for 40 min, which were then poured onto Teflon molds and maintained at 5 ± 0.5 °C for 7 days in order to obtain flat and transparent films with a final thickness of ~0.3 mm. The chemical groups vibrations and molecular configuration of the polymer were analyzed using a Raman spectrometer  equipped with a near infrared laser (λ 785 nm and 1 μm diameter). In order to avoid sample burning, the power of the laser was set to 70 mW. All spectra were acquired using a diffraction grating with a groove density of 600 gmm−1). An empty pan was used as reference. All experiments followed the protocol of cooling down the samples from 25 °C to −10 °C at 2 °C min−1. Measurements were carried out using six replicates. The gelling temperature (Tgelling) of all SG samples was determined from the onset of the exothermic peak observed on the cooling scans. The energy associated to the coil to helix transitions (gelation energy) was defined as the change in enthalpy (ΔH) and it was calculated from the area under the exotherm curve and expressed based on the dry mass of gelatin. The weight of all pans was checked before and after the measurements to ascertain that no water loss occurred during analysis.SG suspensions (10% w/v) were prepared in distilled water under stirring at 60 °C for 40 min. The samples (70 μL) were loaded into aluminum DSC pans (100 μL), hermetically sealed and subjected to thermal scans using a DSC-1 instrument . Prior to the measurements, the DSC was calibrated using indium  were measured using a rheometer . Flow curve measurements were carried out using a cone geometry , whereas oscillatory tests were performed using a flat plate geometry . During analysis, the samples were covered with a solvent trap to avoid water evaporation. Data analysis was conducted using the TRIOS software package . All analysis was carried out using at least five replicates.−1. The range of linear viscosity values for each gelatin was obtained from the viscosity vs. shear rate plot.The flow behavior of the SG samples produced was determined at 25 °C. The shear rate range used was 0.01–1000 sSteady shear curves: The apparent viscosity of the gelatin suspensions was measured as a function of temperature from 40 °C to 0 °C, with cooling rate of 2 °C min−1 and shear rate of 40 s−1. The flow temperature ramps were plotted as apparent viscosity vs. temperature.Dynamic curves: The viscoelastic behavior of the SG suspensions was measured under oscillatory at 1 Hz and 2% strain within the linear viscoelastic range (LVR) at all temperatures measured. The samples were cooled from 40 to 0 °C at 2 °C min−1, and the rheological parameters elastic modulus (G′), loss modulus (G′′), and tan δ (G′′/G′) were determined. Gelling temperature (Tgelling) was associated to the G′′ and G′ intersecting values upon the cooling step.Frequency sweep test: Each SG suspension was cooled at 2 °C min−1 from 40 °C to 5 °C below the gelling temperature obtained in previous section. Then, the temperature was held constant and the response of the SG moduli  to increasing frequency (0.1 to 100 Hz) at a strain 2% was assessed.p ≤ 0.05) among the gelatins extracted under different pH conditions. These analyses were conducted using the software Statgraphics Centurion XVI .When appropriate, one-way analysis of variance (ANOVA) and Tukey test with a 95% confidence level were carried out to statistically assess significant differences (p < 0.05) from 86.9 to 99.4% (dry basis) in SGL and SGH as the processing pH increased from 3 to 5, respectively. Conversely, the non-nitrogenous extract decreased from 12.3 to 0.0% (dry basis) when the pH was increased, in a similar way as reported previously in a work where the effect of pH and the extraction time on structural features of SG were studied [The proximate composition of the SG samples with controlled molecular weight , which were obtained through an extraction method at different pH values, is presented in  studied ,28. More studied .On the other hand, the ash content has been related with mineral content  in the fish scales which are easily dissolved under acidic conditions . Ash conAverage MW of the SG polymer chains was determined by capillary viscometry . SGH preThe MW distribution of the gelatins determined by SDS-PAGE electrophoresis are shown in The amino acidic compositions of SGL, SGM, and SGH with controlled molecular weight are presented in protein in seabass [protein in bovine [The cold water fish gelatin normally have lower hydroxyproline content than warm water fish and mammal gelatins  of SG, which showed no significant differences among the gelatins produced . The pI −1 , 1445 cm−1 (C-H bending), 1240 cm−1 , and 930 cm−1 (helix stretching C-C) [All SG samples were evaluated in terms of their molecular structure and characteristic chemical vibrations . As expeing C-C) . The peaing C-C) ,39,40. T−1, 1778 cm−1, 518 cm−1, and 427 cm−1. The peak at 2900 cm−1 corresponds to the C-H stretching vibration and SGL showed greater intensity compared to SGH  and gelation enthalpy (ΔH) of the SGL, SGM, and SGH samples are presented in nterpart ,44,45.The literature has widely correlated gelatin MW with gelation enthalpy and gel strength features. Gel strength has been usually related with the ability of the gelatin to form sufficient helical structures for network formation and stability. Indeed, a linear correlation has been reported between gel strength and the amount of triple-helices present in gelatin-based materials ,46. In o−1), showing higher viscosity values for SGH. All samples showed two well-defined zones of viscosity-shear rate dependence, with SGL and SGM showing similar behavior. At increasing shear rates from 0.01 s−1 to 3 s−1, both SGL and SGM showed a shear thinning behavior with a continuous decrease of viscosity. This behavior has been explained by a continuous alignment of the highly anisotropic chains in the direction of the shear rate [−1, showed both gelatins reaching a Newtonian plateau, which suggest a dimensional stability related with SG chains forming a firm structure as the viscosity curve approaches infinity on a constant slope [−1), which would be explained by the higher MW and smaller MW distribution in SGH, increasing the probability of chains to join each other, hence promoting the gelatin stabilization by the organization in a firm structure at lower values of shear rate, in a behavior that can also be influenced by the higher amount of imino acids present in SGH.Steady shear viscosity values for SGL, SGM, and SGH samples (measured at 25 °C) are shown in ear rate . The secnt slope . In the Similar findings has been reported by Huang et al.  for gelaThe effect of the controlled molecular weight on the viscosity of the SG samples was evaluated as a function of temperature upon cooling . As expeSGL presented a lower viscosity than SGH. Indeed, at 25 °C viscosities were 0.021 and 0.281 Pa·s, respectively . DiffereThe differences in viscosity at the temperature range tested between the gelatins could also be explained by the observed differences in MW distribution, average MW, and possible number of triple helical structures, as has been extensively reported in the literature ,31,44,52Temperature sweeps under oscillatory conditions at constant strain reporting the storage and loss moduli  of SGL, SGM, and SGH are depicted in The viscoelasticity of the gels of SGL, SGM, and SGH by oscillatory frequency sweeps assessed at 5 °C below the gelling temperature are presented in The fact that GS behaves as strong gels in terms of viscoelasticity for the MW and MW distribution tested, strongly supports and encourages the potential uses of SG in biomedical applications. Specifically, in terms of tissue engineering the performance of a GS-based construct developed by 3D printing or analogous technologies, could be complementarily explained by the viscoelastic response of gelatin. Thus, despite of the lower stiffness showed by SG over mammal gelatins reported by Zaupa et al.  and its This study showed the direct correlation existing between MW and molecular structure of SG with macromolecular properties such as the gelation energy and rheological performance. The high molecular weight of salmon gelatin (SGH) was directly correlated with the highest viscosity, gelling temperature, and G′. Raman spectra and gelation energy suggest a higher amount of helical structures in this SG sample. Conversely, SGL showed lower viscosity and G′, most possibly because of the hydrolytical conditions addressed during sample preparation allowed the rupture of elemental chains resulting in low MW chains with less capability to organize forming helical structures.Interestingly, despite the low gelling temperature and low viscosity which allow to SG to flow at room temperature, it behaved as a strong gel in terms of viscoelasticity once it is structured in hydrogels. Although our study showed clear differences in G′ among SG samples assessed by the frequency sweep test, the strong gel condition resulted to be independent of MW tested and molecular configuration because it was observed in all three gelatin samples tested, even in SGL.Therefore, our study highlights the role of MW and molecular configuration for structuring gelatin-based matrices and thereafter with the performance of this material at the macroscale. We believed this analytical approach is valuable in providing further information on the structure-function relationship, especially on GS and potentially on other gelatins from cold-adapted fish. Hence, our study suggests the feasibility in defining the thermal and rheological properties of SG throughout tuning by MW through extraction processing conditions.These results open interesting perspectives for the design of low viscosity gelatin-based materials with improved properties to be required for applications in any high-value applications that requires fine structuring using low viscosity materials such as electrospinning, microfluidics, micropatterning, and ink-jet bioprinting.


In this paper, we present a targeted PCa imaging strategy using a real-time molecular photoacoustic imaging system integrated with a handheld US probe (PAI/US) and synthesized an integrin To prepare cRGD–ICG, ICG-NHS was linked to cRGD through carboxyl-co-reaction. In vitro PA imaging ability of cRGD–ICG was tested. Orthotopic PCa-bearing rats were used as animal models. After injected with either cRGD–ICG or non-targeted probe, rats were implemented with PA imaging to confirm the specific accumulation of cRGD–ICG at tumor region. Moreover, pathological frozen slices were made to observe distribution of the probe in prostate tissue ex vivo.P < 0.05). Significantly higher cRGD-ICG accumulation was observed under confocal microscopy in the tumor region than in normal prostate tissue.A small molecular PAI probe was synthesized and exhibited excellent targeted imaging ability in vitro. In vivo photoacoustic imaging was carried out after intravenous injection of cRGD-ICG in orthotopic PCa-bearing rats under the facilitation of the PAI/US system. Maximum molecular photoacoustic signals were observed in the tumor area in vivo after the probe injection, which showed 3.8-fold higher signal enhancement than that in the control group (All our results showed that the comprehensive strategy provided a high-yield and reliable method for PCa diagnosis and targeted prostate biopsy, with great clinical translation potential. Prostate cancer (PCa) is the most common malignancy and is the third highest cause of death among the tumor-bearing male population in the united states . UniversUS molecular imaging is also a complementary tool to enhance the accuracy of PCa diagnosis while using US. A first-in-human US molecular imaging trial with BR55 as a vascular endothelial growth factor receptor 2-targeted contrast agent has shown promise for the specific detection of human PCa . HoweverPhotoacoustic imaging (PAI) is an emerging clinical modality that combines a high sensitivity inherent to optical properties, as deep as 5 cm, and a high spatial resolution inherent to US imaging . The phoFor molecular PAI, exogenous contrast agents including nanoparticles and dyes are commonly used to provide sufficient contrast in the signal. Specifically, the small molecular dye indocyanine green (ICG) has great translational potential for clinical PAI because of its high biosafety level. ICG is a near-infrared fluorescent dye already approved by the US Food and Drug Administration (FDA) for clinical imaging applications . Consequαvβ3 which interacts with RGD motifs of the extracellular matrix is known to play a critical role in tumor growth, angiogenesis and metastasis in several tumor types including PCa by modulating cell adhesion, proliferation and migration [αvβ3 integrin receptor would enable noninvasive monitoring of tumor angiogenesis and metastasis using PAI [The cell adhesion molecule integrin igration , 17. Molsing PAI . This ICIn the current study, we present a photoacoustic molecular imaging-based technique to identify malignancies by enhancing both functional and molecular information of PCa using a synthesized molecular probe to increase the detectability of prostate cancer for better US-based targeted prostate biopsy, overcoming the limitations of previous targeted methods.Monomeric cyclic RGD peptide c(RGDyK) was used  for the synthesis of the probe. Appropriate c(RGDyK) was mixed with ICG-NHS ester  in a molar ratio of 1:3 by dissolving in dimethyl sulfoxide (DMSO). The reaction mixture was incubated for 4 h in the dark at room temperature. Subsequently, the mixture was diluted to 3 ml by adding water. The solution was then transferred to a dialysis device for 48 h at 4 °C to remove free ICG. The concentrated c(RGDyK)-ICG solution was subsequently lyophilized to remove organic solvents. Finally, the powder of c(RGDyK)-ICG was dissolved in water and stored at − 20 °C in the dark until use. Brief procedures describing the cRGD-ICG synthesis are shown in Fig. Ultraviolet-visible (UV-Vis) spectra and fluorescence spectra of the probes were obtained by using a UV-Vis spectrophotometer  and a fluorescence spectrophotometer , respectively. In vitro photoacoustic imaging of the probes was performed using a handheld US linear array-based photoacoustic imaging system . A series of different concentrations of probes (1.25–20 μg/ml) were placed in 96-well plates by adding 200 μl of solution to each well. Further quantitative photoacoustic signals of the probe were obtained using MATLAB software.αvβ3 integrin and human embryonic kidney cells (293 T) barely expressing integrin αvβ3 were permitted and purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences . Both cell lines were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. When the cells reached 70% confluence, they were trypsinized and subcultured. All cell culture reagents were obtained from Invitrogen Corporation . Cells were seeded in 6-well plates at a density of 1 × 105 cells per well and were incubated overnight in complete cell growth medium. The 0.5 ml medium containing either cRGD-ICG or cRAD-ICG was incubated with the cells for 1 h at 37 °C. The medium was then changed back to complete cell growth medium for normal cell culture before conducting binding affinity experiments.Human umbilical vein endothelial cells (HUVECs) expressing To examine the intracellular uptake of cRGD-ICG or cRAD-ICG, both HUVECs and 293 T cells were incubated with either cRGD-ICG or cRAD-ICG (20 μg/ml) for 1 h at 37 °C. After incubation, unbound dyes were removed with a gentle PBS wash, and the medium was replaced before imaging. With the obtained final solution, flow cytometry  was performed to determine quantitative binding through the ICG signal.Cells also underwent a similar procedure to test the cellular targeting ability of PAI. Once cell solutions were collected, they were subcutaneously injected into the backside of a normal murine model and were observed with a photoacoustic system . Further quantitative data were obtained offline using MATLAB software.n = 6 in each group). The rats were fasted 18 h before the operation but were fed chow and water otherwise. The experiment began after the rats were appropriately anesthetized by injecting pentobarbital sodium . Then, longitudinal incisions were made in the lower abdomen to expose the prostate. A 50 μl human prostatic carcinoma cell line (PC-3) suspension at 5 × 106/ml was then injected into the gland. The incision in the abdomen was closed, and successful orthotopic PCa modeling was confirmed 3 days after the operation by pathology .All animal experiments were approved by the Sun Yat-sen University Animal Care Committee. All experimental rats were purchased from Charles River . A male nude rat (Sprague-Dawley rat) bearing an orthotopic xenografted PCa model was established. The operation procedures were as follows: 12 mature rats weighing 250–300 g were used for the study. All rats were kept in a temperature- and humidity-controlled room and were randomly divided into 2 groups: the cRGD-ICG and cRAD-ICG groups  solution via tail vein. Imaging was performed at a series of time points after injection: 0 h, 5 min, 2 h, 4 h, 8 h, and 12 h. We used the PAI/US dual-modality imaging system with the following details: 7 MHz linear array transducer  with 128-element (pitch 3 mm), lateral and axial resolution of 336 and 235 μm, respectively, 15 mJ/cmications , 20. Theαvβ3-targeted PAI signal using a MATLAB-based algorithm. The engineer was blinded to group assignment and outcome. The coregistered B-mode US images were used as guidance to select the region of interest (ROI) in the prostate gland comprising both normal and cancerous tissues. Within the identified ROI, PAI signals identifying PCa tissues were analyzed. The quantitative PAI signal was presented as the ratio of pixels containing molecular signal postinjection compared to the signal preinjection. In addition, absolute quantitative PAI signals at each point of imaging depth were calculated.We quantified the integrin Twelve hours postinjection, animals were euthanized by injecting excessive pentobarbital sodium 100 ~ 150 mg/kg. Afterwards, the whole prostate including the tumor was resected and frozen at the optimal cutting temperature (O.C.T.) for pathology . A confocal laser scanning microscope imager  was used to acquire fluorescent images of tissue sections. The ICG component in the molecular probe can be seen in photoacoustic images in vivo as well as in fluorescence images of tissue sections. The pathology of suspicious accumulation of the cRGD-ICG region, identified as PCa in real-time PAI imaging, was confirmed by the pathology results. Tissue sections (10 μm thickness) of the two groups were rinsed in PBS for 5 min to remove the O.C.T. compound and then fixed for 5 min in 4% PFA. Slides were then imaged using PA at 405 nm for the nuclei and 633 nm for ICG.P-value of less than 0.05 was considered indicative of a statistically significant difference in values.All experiments were repeated 3 times, and the data were analyzed using Student’s t-tests and one-way ANOVA . Unless otherwise specified, data were expressed as the means ± standard deviation. A two-sided ε-amino group of the lysine residue of the RGD peptides.As described before, brief procedures of c(RGDyK)- ICG synthesis by amide linkage are shown in Fig. In buffer, as shown in Fig. αvβ3 receptor in vitro, we tested both the HUVEC cell line and 293 T cell line (as a control). After incubating 50% of HUVECs with a targeted agent (cRGD-ICG) and another 50% with a nontargeted agent (cRAD-ICG), cells were collected for flow cytometry. As shown in Fig. αvβ3 receptors exhibited much stronger ICG signals when incubated with cRGD-ICG than when incubated with cRAD-ICG. The quantitative analysis showed that the binding efficacy of cRGD-ICG towards HUVECs was 93.8 ± 5.7% (P < 0.05), which confirmed the excellent targeting ability of cRGD-ICG, laying the foundation for in vivo molecular imaging. In the control cell groups, 293 T cells with a very low expression of integrin αvβ3 receptors barely displayed ICG signals when incubated with cRGD-ICG or cRAD-ICG. In addition, the difference in cellular uptake for 293 T cells was not statistically significant between cRGD-ICG and cRAD-ICG (P > 0.05).To examine the binding affinity of cRGD-based probes towards the integrin P < 0.05). This result demonstrates the targeting ability of cRGD-ICG at the cellular level for enhanced PAI imaging.We then conducted live-cell PAI using the same cell lines to verify the cellular targeting ability. Cells incubated with probes were injected subcutaneously into the dorsal side of a murine model to facilitate PAI. As shown in Fig. αvβ3 are shown in Fig. αvβ3 molecular probe for murine orthotopic PCa targeting, cRGD-ICG was injected into 6 rats via the tail vein. cRAD-ICG was injected into the remaining 6 rats and was used as a negative control probe. PAI was performed at a series of time points before and after probe injection to better understand the physiological behavior of cRGD-ICG. PAI imaging of the abdominal region of the murine models was performed with rats laying on their dorsal size and the transducer placed anteriorly. The maximum amplitude projection images of one of the rat’s abdomens are shown in Fig. The successful murine orthotopic prostate tumor model is shown in Fig. αvβ3 molecular signals merged with B-mode US images, providing valuable anatomical information around the tumor. The rats injected with cRGD-ICG showed significant molecular PAI signal enhancement at the orthotopic tumor area 12 h postinjection, with US showing the surrounding tumor structures  compared to the baseline preinjection signal  to identify PCa. As shown in Fig. res Fig. a. In theres Fig. a. The prnal Fig. c. The qunal Fig. d.Fig. 6αvβ3 molecular PAI signal was confirmed to accumulate mostly in the tumor region with no significant signal accumulation in the normal prostate region  distributed along tumor neovascularization in the cRGD-ICG group were detected Fig. . This reSystematic biopsy for prostate cancer detection, which is currently the gold standard to evaluate PCa, suffers from several limitations including low accuracy, uneven sampling, which can lead to undersampling of clinically significant prostate cancer under diagnosis, and complications. As presented in the introduction, mpMRI and molecular US imaging are investigated for targeted biopsy to improve the efficiency of the core biopsy but have limitations , 6. DeteThe PAI system integrated with the US system provides such an opportunity to improve the efficacy of prostate biopsy. Photoacoustic molecular imaging compliments US imaging since it enables monitoring of the disease process with additional molecular and functional information in addition to providing anatomical structural information. The PAI is more accurate in distinguishing malignancies, as reported in several studies –10. The In this study, we presented a molecular PAI targeting of PCa using synthesized ICG, an FDA-approved small molecular dye. Targeting substances such as peptides and proteins linked to the dye have been evaluated in the past to achieve targeting ability. In fact, proteins  could achieve higher targeting affinity and specificity than the peptide used in this study but have poorer tissue penetration and clearance due to their larger size, hindering their wider application. Thus, the molecular probe synthesized in this study showed greater clinical translation potential.A few other studies have focused on molecular PAI to detect PCa in vivo. Ni et al. monitored PCa growth in an orthotopic mouse model through 3D US-assisted PAI functional imaging by evaluating the angiogenesis feature of the tumor instead of molecular information . Two simIn this study, we also demonstrated the depth of penetration advantage of PAI in the rat orthotopic PCa model. Even with a certain distance between the skin and the tumor area, excellent molecular PAI images were successfully obtained for the whole prostate gland. The molecular PAI signal in the cRGD-ICG group peaked at a depth of 10 mm, where the orthotopic tumor was located, demonstrating the targeting ability of the probe. In fact, the depth of penetration of PAI is more than 5 cm, which meets basic clinical needs.P < 0.05) PA signal in the tumor region than that of rats injected with the nontargeted probe, which validates the significant targeting ability of our molecular PAI for PCa. The merged imaging method enabled visualizing and quantifying tumor malignancy at a molecular level and increased the sensitivity of diagnosing the tumor, which will potentially increase the accuracy of targeted biopsy and even local treatment for PCa clinically.In the present study, PAI and US imaging were merged to copresent orthotopic PCa and the surrounding anatomical area, including the prostate and bladder. This anatomical information is vital not only for avoiding injury to normal tissues during biopsy procedures but also for identifying PCa areas in PAI. The merged PAI and US images identified PCa for targeted biopsy, aided by the critically important targeting ability of the molecular probe towards tumor angiogenesis. We quantitatively analyzed the molecular imaging results of PCa in this study. The rats injected with the targeted probe showed a 3.8-fold higher . Employed with cRGD-ICG, molecular PAI showed excellent diagnostic ability for murine orthotopic PCa models by PAI/US merged imaging. The strategy that we developed in this work showed great potential for guiding targeted prostate biopsy towards highly suspicious regions, thus increasing the biopsy rate during systematic biopsy. Because the dye is already FDA approved and the PAI depth of imaging is more than 5 cm, we believe that our study presents a compelling reason to use our system in real clinical situations. In the near future, we plan to conduct a clinical study in humans.In this study, we successfully synthesized an integrin Additional file 1: Figure S1. Modeling of rats with orthotopic prostate cancer. A. Ultrasound images. B. General observation. Blue arrow: bladder. Yellow arrow: tumor. C. Observation under the microscope. Black arrow: prostate gland. Red arrow: cancerous tissue. (Magnitude: 50 ×). Figure S2. Pathology including both H&E staining and immunochemistry staining of both prostate gland and tumor tissue. (Magnitude: 100 ×). Figure S3. Representative PA map images of rats with orthotopic PCa.


Vibrio vulnificus infection is a rare but fatal foodborne illness. Here, we report a case of Vibrio vulnificus peritonitis followed by severe septicemia in a patient undergoing continuous ambulatory peritoneal dialysis (CAPD) who was treated with hemoperfusion using polymyxin B immobilized fiber.Vibrio vulnificus as the most likely causative pathogen. Hemoperfusion with polymyxin B immobilized fiber was performed to control gram-negative bacterial septicemia with antibiotics targeting the pathogenic organism. The patient recovered completely and was discharged without complications.A 63-year-old man undergoing CAPD was admitted to the emergency room due to general weakness, fever, and abdominal pain with hazy dialysate. Two days before admission, he had eaten raw fish. Initial laboratory tests including peritoneal fluid analysis suggested peritonitis. Despite empirical intraperitoneal antibiotic treatment, his fever did not subside, and multiple vesicles on the extremities newly appeared. The result of initial peritoneal fluid culture and blood cultures reported Vibrio vulnificus infection in susceptible immunocompromised patients is important for early diagnosis and prompt management. Peritonitis should be noted as a clinical manifestation of Vibrio vulnificus infection in CAPD patients, and polymyxin B hemoperfusion along with proper antibiotics could be considered as a treatment option.Suspicion of  Vibrio vulnificus is a gram-negative rod-shaped bacterium found in warm marine environments. The main clinical manifestations of Vibrio vulnificus infection are gastrointestinal illness, wound infection, and primary septicemia. In particular, oral ingestion of or wound exposure to raw sea products in immunocompromised individuals, such as patients with liver cirrhosis, iron overload disorders, diabetes mellitus, steroid use, and chronic kidney disease (CKD), can lead to sepsis and fatal outcomes [Vibrio vulnificus in a patient undergoing continuous ambulatory peritoneal dialysis (CAPD) who was successfully treated by antibiotics and hemoperfusion using polymyxin B immobilized fiber.outcomes , 2. Even2) filter set, which contain a polyacrylonitrile AN 69 membrane (Gambro). Blood flow rate was started at 100 mL/minute and gradually increased to 150 mL/minute. Effluent volume was set to achieve a clearance of 40 mL/kg/hour. Replacement and dialysate volumes were set using the 1:1 balanced-predilution method. On the hospital day 3, the preliminary result of the initial peritoneal fluid culture reported Vibrio vulnificus as the most likely causative pathogen. Considering the preliminary culture study report, systemic intravenous ceftriaxone (2000 mg twice a day) and oral doxycycline (100 mg twice a day) were initiated, and the intraperitoneal antibiotics were immediately changed to ciprofloxacin (200 mg per each PD). With the change of antibiotics, hemoperfusion with polymyxin B immobilized fiber  was concomitantly initiated, since gram-negative bacterial infection seemed to be the cause of the patient’s hemodynamic instability. In addition to CRRT, 2 hours of hemoperfusion was applied daily for two consecutive days. Approximately 3 h after hemoperfusion commencement, the patient’s electrolyte imbalance and metabolic acidosis were ameliorated, and his blood pressure stabilized, allowing a decrease in the dose of the inotropic agents without compromising his hemodynamic stability. Although the skin lesions on both lower extremities showed slight signs of aggravation, the patient did not exhibit any signs of compartment syndrome, such as motor weakness, altered sensation, or pulselessness. Based on these findings, the orthopedic surgeons decided that it was not necessary to have the patient undergo emergent fasciotomy. Therefore, only sterile dressings on the wet wounds were carried out daily during the ICU stay. The final reports of the bacterial cultures performed at the time of admission confirmed the growth of Vibrio vulnificus from both the peritoneal fluid and the peripheral blood with sensitivity to cefotaxime, ceftazidime, ciprofloxacin, and tetracycline. On hospital day 5, the WBC count in the peritoneal fluid decreased to 57/μL with 3% of PMN cells, and the CRP level decreased from 451.3 to 301.7 mg/L. CRRT was discontinued on the hospital day 6, and the patient was sent to the general ward on the day after CRRT discontinuation. Treatment with antibiotics, including intravenous ceftriaxone and oral doxycycline, was continued for two more weeks. Peritoneal dialysis was continued until hospital day 11 on which the PD catheter was removed. The patient recovered without significant complications and was discharged on the 46th day of hospitalization. The time sequence of key events is presented in Fig. A 63-year-old man who had undergone CAPD for 11 years was admitted to the emergency room due to general weakness, fever, and abdominal pain with hazy dialysate. He was an East Asian male, an office worker, who was 1 meter and 70 centimeters tall weighing 67.0 kg. His CAPD regimen consisted of a, 4 times a day, conventional 1.5% glucose anhydrous based dialysis solution containing lactate. The patient had been anuric for several years. Erythropoiesis-stimulating agents nor intravenous iron replacement had not been prescribed during the last month. However, he had been constantly receiving oral iron replacement. Two days before admission, he ate a sliced raw skate (thornback ray), and abdominal pain and diarrhea developed on the next day. The underlying cause of his end-stage renal disease (ESRD) was chronic glomerulonephritis, and there was no history of liver disease, diabetes mellitus, or steroid use. On admission, his body temperature was 39.5 °C, blood pressure 116/62 mmHg, and pulse rate 99 beats/min. There was direct and rebound tenderness on the whole abdomen. No definite skin lesions were discovered on any part of his body at initial evaluation, and the exit site of the peritoneal dialysis catheter was clean. Serum laboratory findings were as follows: white blood cell (WBC) count 1930/μL, hemoglobin level 8.0 g/dL, aspartate/alanine transaminase levels 37/72 IU/L, serum iron level 36.0 μg/dL, transferrin saturation 22.0%, ferritin level 745.6 μg/L, C-reactive protein (CRP) level 109 mg/L, and procalcitonin level 65.89 ng/mL. Peritoneal fluid analysis revealed that WBC count was 3400/μL with 80.1% of PMN cells. The laboratory results are summarized in Table Vibrio vulnificus is an opportunistic pathogen belonging to the marine environment. It is usually transmitted by ingestion of infected seafood, especially filter-feeding aquatic organisms such as oysters, mussels, and clams. The incidence of Vibrio vulnificus infection has been recently increasing [creasing . Wound iVibrio vulnificus infection [Vibrio vulnificus infection. Dysfunctions of neutrophils, monocytes, and lymphocytes observed in uremia could play a role in increasing the risk of certain life-threatening infections in these patients [Vibrio vulnificus is known to grow rapidly in iron rich environments, a situation commonly encountered among dialysis patients who frequently need blood transfusions or iron supplementation to correct anemia [Several predisposing medical conditions are known to be associated with a high susceptibility to nfection . Renal dpatients . Moreovet anemia , 7.Vibrio vulnificus infection [Vibrio vulnificus [The combination of tetracycline and a third-generation cephalosporin is recommended as the initial antibiotics of choice for nfection . In a prlnificus . AccordiVibrio vulnificus arises from its capsular polysaccharide, cytolysin, and lipopolysaccharides [There have been several attempts to verify the efficacy of hemoperfusion using polymyxin B immobilized fiber in patients with septic conditions. Even though the preliminary results of the EUPHAS  trial demonstrated that polymyxin B hemoperfusion added to conventional therapy significantly improved hemodynamics and organ dysfunction and reduced 28-day mortality in patients with severe sepsis or septic shock who underwent emergency surgery for intra-abdominal infections , the EUPcharides . Since tcharides ), hemopeIn the current case, CVVHDF (continuous venovenous hemodiafiltration) was selected for renal replacement therapy in addition to CAPD due to the rapid exacerbation of hyperkalemia and metabolic acidosis. Continuous equilibrated peritoneal dialysis (CEPD) or high-volume peritoneal dialysis (HVPD) could also be considerable options in sepsis patients who are in hypercatabolic states. However, in the current case, abdominal distension and ileus was aggravating which prevented the application of high volume peritoneal dialysis treatments. The decision to commence CRRT and obtain vascular access also enabled the rapid administration of hemoperfusion.Vibrio vulnificus [Vibrio vulnificus peritonitis followed by severe septicemia and necrotizing fasciitis in a patient undergoing CAPD [Vibrio vulnificus which were successfully treated by polymyxin B hemoperfusion [Vibrio vulnificus followed by septic shock was effectively treated with proper antibiotics plus the use of polymyxin B hemoperfusion. With the deterioration of the septic shock, leukopenia was observed temporarily. This transient finding was thought to be caused by the fulminant infection. Several previous reports have also shown Vibrio vulnificus infection to be accompanied with leukopenia. The presence of leukopenia in these patients was found to be associated with poor prognosis [To date, there have been two reported cases of CAPD peritonitis caused by lnificus , 16 and ing CAPD , all of erfusion , 18. Therognosis , 20. In Vibrio vulnificus sepsis in PD patients treated with a combination of ciprofloxacin and third generation cephalosporin without hemoperfusion. However, although these reports do not specify the time interval between antibiotics treatment and blood pressure stabilization, they do describe the period to clinical improvement to be in the range from few days to weeks compared to the several hours of the current case. Secondly, the actual endotoxin levels before and after hemoperfusion treatment were not available. Unfortunately, assays measuring the amount of endotoxin such as EAA (Endotoxin Activity Assay) are not yet available in Korea and hemoperfusion is prescribed empirically in hemodynamically unstable gram-negative sepsis patients.There are several limitations of this case report. First, the main reason for the initial hemodynamic stabilization cannot be completely specified. Hemoperfusion had been initiated with concurrent change of antibiotics in the present case. Since stabilization of hemodynamics had been observed after a relatively short time after antibiotics regimen change and hemoperfusion administration, it could be assumed that hemoperfusion may have played a bigger role compared to the changed antibiotics. Previous reports do report cases of Vibrio vulnificus infection in susceptible immunocompromised patients are essential for early diagnosis and proper management, which can dramatically improve clinical outcomes. It would be worthwhile to acknowledge that peritonitis can be an early clinical sign of Vibrio vulnificus infection in CAPD patients, and polymyxin B hemoperfusion should be recognized as a treatment option in such patients.In conclusion, thorough history taking and suspicion of


Escherichia coli revealed that M4Lys alone caused rapid cell lysis. Treatment of E. coli with a Sec inhibitor did not inhibit the lysis activity of M4Lys, indicating that the Sec pathway is not involved in M4Lys-mediated cell lysis. Truncation of the TMD eliminated the cell lysis phenomenon, while production of the TMD alone did not induce the cell lysis. All these findings demonstrate that M4Lys is a novel endolysin that has a unique mosaic structure distinct from other canonical endolysins and the TMD plays a critical role in M4Lys-mediated in vitro cell lysis.Most double-stranded (ds) DNA phages utilize holin proteins to secrete endolysin for host peptidoglycan lysis. In contrast, several holin-independent endolysins with secretion sequences or signal-arrest-release (SAR) sequences are secreted via the Sec pathway. In this study, we characterized a novel lysis protein (M4Lys) encoded by the dsDNA phage BSPM4, whose lysis function is not dependent on either holin or the Sec pathway in vitro. In silico analysis of M4Lys revealed that it contains a putative virion protein domain and an unusual C-terminal transmembrane domain (TMD). Turbidity reduction assays and liquid chromatography-mass spectrometry using purified peptidoglycan showed that the virion protein domain of M4Lys has peptidoglycan lysis activity. In vitro overproduction of M4Lys in  Bacteriophages rely on diverse cell lysis mechanisms to release their progeny from host bacteria. Bacterial lysis by phages can be accomplished in at least two different ways, i.e., peptidoglycan degradation by endolysins or the inhibition of peptidoglycan synthesis ,2,3. TheEndolysins are peptidoglycan hydrolases that disintegrate integrity of the cell wall by cleaving the peptidoglycan. It is known that endolysins generally cleave glycosidic bonds, peptide bonds, or amide bonds of the peptidoglycan. Holins are small membrane proteins that dissipate membrane potential or make holes in the membrane to allow endolysins to access the peptidoglycan target ,6. In thOenococcus oeni phage fOg44 (Lys44) contains a typical signal sequence that is converted into the active form after cleavage of the N-terminal signal peptide. The N-terminal region of Lys44 functions as an export signal for the endolysin to translocate to the periplasmic region  were used if necessary. When indicated, isopropyl-β-thiogalactopyranoside (IPTG) and sodium azide (NaN3) were added at 0.1 mM and 1-10 mM, respectively.The bacterial strains and plasmids used in this study are listed in E. coli BL21 (DE3) strain and induced to examine cell lysis kinetics. The putative holin gene  was PCR-amplified with the BSPM4Holin-F-NcoI and BSPM4Holin-R-HindIII primers and cloned into the pETDuet-1 as described above. To coexpress M4Lys and M4Holin, both genes were cloned into pETDuet-1 using the primers above. To produce the proteins, 0.1 mM of IPTG was added 3 h after incubation and the lysis kinetics of each strain was observed as previously described [E. coli BL21 (DE3) strain was used as a control. All the primers used for PCR amplification are listed in The putative endolysin gene ORF_38, M4Lys) was amplified by polymerase chain reaction (PCR) with the BSPM4Lys-F-NdeI and BSPM4Lys-R-XhoI primers using phage BSPM4 DNA as template. The PCR product was digested with the respective restriction enzymes and cloned into the pETDuet-1 vector  and sequenced. The recombinant plasmids were, then, used to transform , M4Lys wE. coli BL21 (DE3) strain was incubated for 2 h until an optical density at 600 nm (OD600) of 0.5 was reached and was, then, supplemented with 0.5 mM IPTG to produce the M4LysΔTMD protein. After incubation for 3 h, cells were suspended in lysis buffer  and sonicated  to break bacterial cell walls. Sonicated cells were centrifuged at 15,000× g for 20 min to obtain the supernatant containing soluble proteins. His-tagged M4LysΔTMD was purified using a Ni-nitrilotriacetic acid Superflow column  according to the manufacturer’s instructions. The purified protein was visualized with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and stored in storage buffer  after exchanging buffers using a PD Miditrap G-25 column  [To purify a soluble form of M4Lys protein lacking the C-terminal TMD (designated as M4LysΔTMD), the M4LysΔTMD coding gene was cloned into the pET28a vector using the BSPM4Lys-F-NdeI and BSPM4Lys2-R-XhoI primers. In the cloning experiments to generate recombinant plasmids, DNA fragments encoding the putative endolysin gene were inserted between the NdeI and XhoI restriction sites of the pET28a plasmid resulting in an N-terminal His-tag. The recombinant plasmid-harboring cks, UK) .E. coli peptidoglycan (muropeptides) as substrates. Crude muropeptides of E. coli BL21 (DE3) were extracted as described previously with slight modifications [E. coli cells were harvested and the cells were disrupted by sonication. The cell suspension was then centrifuged at 1400× g for 10 min to obtain the crude cell wall lysate. Muropeptides were pelleted by centrifugation at 27,000× g for 5 min, resuspended in 4% SDS solution, and boiled for 10 min. Muropeptides were washed using cold distilled water at least 3 times, suspended in Tris-HCl buffer (pH 8.0), and stored at 4 °C until use. Peptidoglycan lysis activity of M4LysΔTMD was examined by measuring a reduction in OD600 of the muropeptide solution [600 was measured using a SpectraMax i3x  microplate reader. The reduction of turbidity was calculated by comparing with that of the respective control group without enzyme treatment. The bacterial strains used for host range analysis are listed in S. Typhimurium LT2C [Lytic activity of purified M4LysΔTMD was determined using purified ications ,21. In bium LT2C .E. coli MG1655 cells were grown exponentially (OD578 = 0.6) and harvested by centrifugation at 12,000× g for 15 min at 4 °C. The cell pellet was resuspended in 3 mL of ice-cold deionized water (D.W.) and 3 mL of boiling 8% SDS was added dropwise with vigorous stirring within 10 min. The mixture was boiled for 30 min and incubated overnight at room temperature. Muropeptides were collected by ultracentrifugation . The pellet was washed 4 times with 8 mL of D.W. to remove residual SDS. High molecular weight glycogen and covalently bound lipoprotein were cleaved by adding α-amylase  and pronase , respectively. Samples were mixed with an equal volume (8 mL) of 8% SDS and boiled for 15 min to inactivate the enzymes. Muropeptides were collected by ultracentrifugation and washed 3 times with D.W. to remove SDS from the pellet. The pellet was suspended in 20 mM Tris-HCl buffer (pH 8.0) and stored at 4 °C until use.Muropeptides for HPLC analysis was prepared as described above with slight modifications . Brieflyg, 15 min at 20 °C) and treated with purified M4LysΔTMD at 25 °C overnight. Samples were heat-inactivated for 10 min and reduced with 5 mg sodium borohydride in 500 mM sodium borate buffer (pH 9.0) for 30 min at room temperature. Borohydride was inactivated by adding 20% phosphoric acid, the pH adjusted to 4, and the sample filtrated using a 0.2 μM filter. Prepared samples were loaded onto a Kinetex C18 column  and a UHPLC C18 column  was used as a guard column. RP-HPLC was performed using the Agilent Technologies HPLC 1260 system at the Korea Basic Science Institute . Samples were injected onto a column preheated to 50 °C and allowed to bind at a flow rate of 0.5 mL/min with a solution of 1% acetonitrile and 0.1% trifluoroacetic acid (TFA) for 10 min. A gradient of 1% to 10% acetonitrile in 0.1% TFA solution was used for elution at a flow rate of 0.5 mL/min for the next 60 min. Absorbance was detected at 205 nm and the fractionated muropeptides were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis was performed as previously described  with a f1–224, BSPM4Lys-F-NdeI and BSPM4Lys5_4-R-XhoI for M4Lys1–225, and BSPM4Lys-F-NdeI and BSPM4Lys6-R-XhoI for M4Lys1–226, respectively. All three PCR-amplified constructs were cloned into the pET28a vector and were transformed into E. coli BL21 (DE3) cells. Then, recombinant plasmid-harboring E. coli cells were used for cell lysis tests with 0.1 mM IPTG induction. Primers used for PCR amplification are listed in To understand the role of M4LysΔTMD and the TMD region, each recombinant gene was PCR-amplified, cloned into the pETDuet-1 dual-expression vector, and expressed with 0.1 mM of IPTG, as described above. The TMD region was truncated from the gene encoding the M4Lys protein with various primers. Primers BSPM4Lys-F-NdeI and BSPM4Lys2-R-XhoI were used to generate the TMD-truncated construct M4LysΔTMD. The following three primer sets were used to generate the partially truncated TMD: BSPM4Lys-F-NdeI and BSPM4Lys5_3-R-XhoI for M4LysP distance values [Phylogenetic trees were constructed based on alignment of the M4Lys protein sequence with the amino acid sequences from 37 Gram-negative bacteriophage endolysins available on the NCBI database. Protein sequences were aligned with the Clustal X2 program  and the e values . Amino ae values ,27.Salmonella phages such as iEPS5 and FSLSP099 [The phage BSPM4 genome sequence includes two putative lysis genes in the predicted lysis cassette, putative lysis protein B (ORF_37) and putative lysis protein A (ORF_38). A hypothetical gene (ORF_39) was identified downstream of the two putative lysis genes A. ArrangFSLSP099 ,29. HoweE. coli expression system. Endolysin is synthesized and accumulated in the cytosol until the holin creates pores in the inner membrane, thus allowing the endolysin to reach its peptidoglycan substrate target [E. coli cells were considerably lysed between 20 and 60 min after putative lysis protein A (M4Lys hereafter) alone was produced by IPTG induction  and the putative lysis protein A (ORF_38) using an e target ,30. Typie target ,31. Howenduction B. Furthenduction B. Coexprnduction B. Overalnduction B. We spenduction B. HoweveMost holin-independent endolysins contain N-terminal signal peptide sequences or SAR sequences essential for their secretion to the periplasm. These sequences often function as secretion sequences for Sec translocase ,10,11. H3, a well-known inhibitor of E. coli Sec translocase [3. Increasing concentrations of NaN3 delayed cell lysis by M4LysA but did not inhibit cell lysis  (data not shown). Since production of the full-length M4Lys protein caused rapid cell lysis  have a globular structure with a single catalytic domain . The NCBll lysis B, we speA putative TMD was predicted in the C-terminus of M4Lys using Phobius, Phyre2, and TMHMM algorithms ,35,36 FA. AnothePeptidoglycan lytic activity of the virion protein domain was assessed by deleting the C-terminal transmembrane domain of M4Lys (M4LysΔTMD). The lytic activity of the full-length M4Lys could not be measured since we failed to purify the protein in spite of repeated attempts, presumably due to the rapid cell lysis that occurred upon its induction. M4LysΔTMD of the predicted size (24.5 kDa) was purified  and incuN-acetylmuramidases (lysozymes), endo-β-N-acetylglucosaminidases (glycosidases), lytic transglycosylases, endopeptidases, and N-acetylmuramoyl L-alanine amidases [E. coli peptidoglycan (muropeptides) was predigested with a muramidase (mutanolysin) and incubated with purified M4LysΔTMD. Then, the muropeptides were fractionated by RP-HPLC and analyzed by MS (N-acetylglucosaminidase (glycosidase) activity of M4LysΔTMD. This result again supports the Phyre2 prediction that displayed structural homology between the N-terminus of M4Lys and the glycoside hydrolase domain of the flagella protein FlgJ from Salmonella. Interestingly, endopeptidase activity of M4Lys could also be inferred from the observation that a crosslinked dimer of disaccharide-tetrapeptide (peak 3) was reduced and a D-Glu-mDAP crosslink cleaved disaccharide  was generated   B.Our current data suggest that the ORF_37 of BSPM4 phage is not required for M4Lys-mediated cell lysis in vitro. However, we cannot currently rule out the possibility that ORF_37 is necessary for optimum cell lysis during BSPM4 phage infection in vivo.Salmonella. Subsequent BLASTp homology searches and amino acid sequence alignments revealed that the sequence of M4Lys is highly conserved (97% to 99% identity) among Chi-like phages  along with those originating from flagella-targeting (Chi-like) phages SPN19, iEPS5, FSLSP088, and Chi A 28,43,,43,44,45e phages B. These bacteria A.Salmonella phages. However, since this study provided indirect, circumstantial evidence on the potential role of TMD in M4Lys function, further studies are warranted to investigate its specific role in cells. Additionally, we hypothesize that the unique structure of M4Lys could be due to the recombination of two domains and further studies are being conducted in our laboratory to examine the interactions of the two M4Lys domains in an attempt to investigate the mechanism behind the formation of the observed mosaic domain.In this study, we identified and characterized the cell lysis characteristics of M4Lys, a novel endolysin that causes cell lysis when overproduced in vitro. Of particular note is the unusual structure of M4Lys that contains a putative virion protein domain and a TMD. A series of cell lysis kinetic analyses suggested that M4Lys has peptidoglycan degradation activity and that TMD is indispensable for host cell lysis by M4Lys when M4Lys is overexpressed in vitro. This finding enhances our understanding of the host cell lysis mechanism, especially by the flagella-targeting


Molecular cytopathology is a rapidly evolving field embracing both conventional microscopy and molecular pathology. Its growing popularity stems from the fact that in many types of advanced cancers, including non small cell lung cancer (NSCLC), cytological samples often constitute the only available specimens for morphomolecular analysis. Indeed, non formalin fixed and paraffin embedded (FFPE) cytological samples feature a higher quality of extracted nucleic acids than histological specimens. However, because of the growing complexity of molecular testing, several efforts should be made to validate the analytical performance of the wide array of currently available molecular technologies, including next generation sequencing (NGS). This technology has the terrific advantage of allowing simultaneous detection of scores of predictive biomarkers even in low-input DNA/RNA specimens. Here, we briefly review the role of the modern cytopathologist in the morphomolecular diagnosing of advanced stage NSCLC and the adoption of NGS in conventional cytopreparations  and supernatants. EGFR), Anaplastic Lymphoma Kinase (ALK), ROS Proto-Oncogene 1 Receptor Tyrosine Kinase (ROS1), and V-Raf murine sarcoma viral oncogene homolog B (BRAF)  are therapeutically advantageous not only in improving overall patients' survival but also in reducing treatment-associated toxicities . HoweverB (BRAF) –5. In adB (BRAF) , 7. DespB (BRAF) , the scaB (BRAF) . DespiteB (BRAF) . In factB (BRAF) . MoreoveB (BRAF) .Interestingly, typical cytopreparations  are not the only alternatives to histological samples. Indeed, several studies have investigated the possibility of using supernatants for molecular analysis. Until recently, supernatants were typically discarded after cytological preparations. Nowadays, instead, they are being used as a valid source of tumoral nucleic acids for molecular analysis, enabling preservation of diagnostic slides .Since different types of cytopreparations are suitable for molecular analysis , the valHere, we briefly review the role of modern cytopathologists in the treatment decision making algorithm of advanced-stage NSCLC patients. Further, we summarize the latest literature on the application of NGS not only to conventional cytopreparations  but also to supernatants.Over the last decade, cytopathology practice has undergone a paradigm shift in the rapidly evolving field of molecular cytopathology. In this fast-changing scenario, modern cytopathologists have made great strides in the “world” of molecular testing, thus ensuring comprehensive management of advanced-stage cancer patients in modern multidisciplinary team (MDT) settings . In thisOverall, molecular cytopathologists are pivotal in the pre-analytical evaluation of cytological samples for molecular analysis . In thisIn the era of precision medicine, molecular analysis on cytological samples has acquired a relevant role in the management of advanced stage NSCLC patients . DiffereAdequacy of cytological samples for NGS analysis is assessed according to tumor cellularity and viability. Both factors may, however, vary depending on the analytical sensitivity of the platform used . As mentAs a general rule, to assess whether a sample is appropriate for NGS analysis, cytopathologists should ascertain that the fraction of tumor cells is equal to or higher than 20% . HoweverEGFR, 33.3% Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), and 7.7% BRAF mutated cases  mutation] were detected (EGFR exon 20 p.S768_D760DUP). Strikingly, all testing was performed without sacrificing any precious diagnostic material , Karnes et al. demonstrated the suitability of FNA lung adenocarcinoma smears for NGS analysis  to NGS analysis to assess the molecular status of clinically relevant biomarkers, we observed that only a few smears  generated inadequate libraries. Indeed, 20.0, 26.4, 4.0, and 0.8% of the samples revealed EGFR, KRAS, BRAF, and Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS) gene mutations, respectively , Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) gene alterations. Of note, real-time polymerase chain reaction (RT-PCR) confirmed all EGFR mutations, whereas the MiSeq platform  confirmed the other detected alterations (Liquid based cytology (LBC) represents a valuable alternative to more conventional cytopreparations to avoid inadequate management of aspirated material by untrained clinicians . Indeed,erations .Another major advantage of this approach is that LBC samples can be used to build quality controls for different NGS platforms to standardize molecular procedures on cytological specimens. To this end, the Molecular Cytopathology Meeting Group, which involves highly specialized molecular cytopathogists from all over the world, developed artificial genomic reference standards in cytocentrifuge/cytospin format to validate the feasibility of using NGS on routine cytopreparations , 50. Forn = 1 EGFR exon 21 p.L858R, n = 1 EGFR exon 19 p.E746_A750del plus EGFR exon 20 p.T790M, and n = 1 KRAS exon 2 p.G12V) (n = 6 newly diagnosed and n = 6 with lung cancer and acquired resistance to TKIs)  . Impress samples . Likewisial FNAs . The posial FNAs . Overallplet PCR . ResultsPrecision medicine, accompanied by novel targeted treatments, has significantly modified the way advanced-stage NSCLC is treated. In this setting, modern molecular pathology, based on traditional morphological pathology, has a pivotal role in the diagnostic algorithm and subsequent treatment decision making for patients with advanced-stage NSCLC. It is in this context that traditional morphology merges with molecular cytopathology. To date, modern cytopathologists and histopathologists are not only the “custodians” of tissue specimens, but also major players in the management and prioritization of tumor material destined for molecular testing . GeneralIn addition, tumor mutational burden (TMB) has emerged as a highly complex and independent predictor of response to immunotherapy. Briefly, TMB is the total number of somatic, coding, base substitutions, and short insertions/deletions (indels) per tumor genome  58, 59), 5958, 5In conclusion, this review briefly evidenced the active role of the modern cytopathologists in ensuring successful NGS analysis of cytological samples and in contributing to treatment decision-making in advanced-stage NSCLC. Moreover, it has highlighted the value of cytological preparations as starting material for NGS analysis. Indeed, not requiring long fixation time and formalin-based fixatives, some of these preparations generate even better DNA/RNA yields than conventional samples. Moreover, the use of supernatants for detection of clinically relevant driver genes of NSCLC, or other types of advanced cancers, enables cytopathologists to preserve unique and precious cytological slide material indispensable for molecular analysis. Therefore, given the complexity of this ever-evolving clinical scenario, we recommend continuous and adequate training programs for modern molecular cytopathologists.PP and GT: conceptualization. PP, UM, and GT: writing—original draft preparation. GT: supervision and project administration. All authors: methodology, software, validation, formal analysis, investigation, resources, data curation, writing—review and editing, and visualization.UM reports personal fees (as speaker bureau or advisor) from Boehringer Ingelheim, AstraZeneca, Roche, MSD, Amgen, and Merck, unrelated to the current work. GT reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer, and Bayer, unrelated to the current work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The products were obtained in ethanolic solution from a reaction between ortho, meta and para derivatives of fluorophenyl isothiocyanate and hydrazine hydrate. This work presents the theoretical Molecular Electrostatic Potential (MEP) and Highest Occupied Molecular Orbital-Lowest Unoccupied Molecular Orbital (HOMO-LUMO) computational data through Gaussview 5.0.9 and Gaussian09 software. Experimental Cole-cole plot for conductivity determination was also illustrated. The present data is important to manipulate the properties of compounds according to the position of a fluorine atom.One-pot synthetic method was adopted to prepare three isomers 4-( Theoretical computational data for both Highest Occupied Molecular Orbital-Lowest Unoccupied Molecular Orbital (HOMO-LUMO) and Molecular Electrostatic Potential (MEP) are presented in Generally, all present data was related with previous reported on crystal structure of 4-thiosemicarbazide compound, a suspension of ortho-fluorophenyl isothiocyanate  with hydrazine hydrate  was refluxed in ethanol (50 ml) for 5 h. The white precipitate formed was filtered and washed with cold ethanol. Finally, the precipitate was recrystallized from hot ethanol, dried and stored in desiccator filled with silica gel.For 4-(meta-fluorophenyl)thiosemicarbazide and 4-(para-fluorophenyl) thiosemicarbazide compounds, meta-fluorophenyl isothiocyanate and para-fluorophenyl isothiocyanate reactants were used instead of ortho-fluorophenyl isothiocyanate.Whereas, for both 4- was selected as basic set due to standard theory level for C, H, N, S and F elements. Furthermore, the Density Functional Theory (DFT) method, named Becke, 3-parameter, Lee-Yang-Parr (B3LYP) was selected as method to interpret Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) analysis in their optimized structures [Optimized structure of all isomers were carried out with GaussView 5.0.9 and Gaussian 09 software package programme ructures . The strEgap), hardness (η), softness (σ) and the global electronegativity (χ) were calculated by using Eqs. (1)–(4) [The red and green colour of the orbital represents the positive and negative phase accordingly. HOMO-LUMO determination and its other several important key factors for conductivity activity which like energy gap (Δ (1)–(4) ,3.(1)ΔEgMolecular Electrostatic Potential (MEP) is useful to visualize both yellow and blue regions indicate the electrophilic and nucleophilic regions, respectively.2.4ortho-fluorophenyl)thiosemicarbazide dissolved in 30 mL of ethanol was added dropwise into the CMC solution. The mixture was left stirring until a homogeneous solution formed. Propylene carbonate (8 wt%) was then added into the mixture. The mixture was casted into the petri dishes and dried in the oven at 60 °C for 14 h to form a thin film. The same procedure was applied for the polymer electrolyte films containing 4-(meta-fluorophenyl)thiosemicarbazide and 4-(para-fluorophenyl)thiosemi- carbazide isomers.In a beaker, 1.0 g of carboxymethyl cellulose (CMC) was added to 20 mL of distilled water and stirred until it was completely dissolved. In the meantime, for a separate solution containing 0.381 g (16 wt%) of 4-(2.5t = thickness of PE film (cm)The polymer electrolyte films were cut into small discs of 2 cm diameter and sandwiched between two stainless steel electrodes under spring pressure. The measurements were carried out at room temperature (303 K). The conductivity of the PE films was calculated as in Eq. (5) ,8.(5)σ=tA = surface contact area of PE film (cm2)bR = bulk resistance of PE film (Ω)The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Concentrated collagen hydrolysate (HC10CC), rabbit collagen glue (RCG), and keratin hydrolysate (KH) were investigated in terms of their extraction from mammalian by-products and processing by electrospinning. The electrospun nanofibers were characterized by scanning electron microscopy coupled with the energy dispersive X-ray spectroscopy (SEM/EDS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), and indentation tests. The cytotoxicity of the electrospun nanofibers was conducted on L929 fibroblast cells using MTT and LDH assays and cell morphology observations. The electrospun RCG and KH nanofibers morphology showed an average size of nanofibers ranging between 44 and 410 nm, while the electrospun HC10CC nanofibers exhibited higher sizes. The ATR-FTIR spectra performed both on extracted proteins and electrospun nanofibers showed that the triple helix structure of collagen is partially preserved. The results were in agreement with the circular dichroism analysis for protein extracts. Furthermore, the viscoelastic properties of electrospun KH nanofibers were superior to those of electrospun RCG nanofibers. Based on both in vitro quantitative and qualitative analysis, the electrospun nanofibers were not cytotoxic, inducing a healthy cellular response. The results of new electrospun protein-based nanofibers may be useful for further research on bioactive properties of these nanofibers for tissue engineering. The research on the biocompatible natural polymers processing by electrospinning for biomedical applications has increased in recent years ,2. Nanofɑ-chain) and ϒ (trimer of ɑ-chain) in gelatin state [Collagen and keratin are the most valuable natural biomaterials due to their chemical versatility and biological performance. They mimic the extracellular matrices (ECM) of tissues and organs, which consist in a complex composite of fibrous proteins such as collagen and fibronectin, glycoproteins, proteoglycans, and soluble proteins . In order to be designed as extracellular matrix of tissues, the mechanical rheology and the ability of protein to bind with other biopolymers need to be proved. Collagen is a natural biopolymer, the most abundant protein in mammals, which is widely used in bone restoration engineering , processin state . Keratinin state . Keratinin state .The most difficult steps in the electrospinning process are related to the dispersion of proteins molecules in proper solvents or polymer solutions so that the triple helix structure is preserved . Low conw/v, 25% w/v, and 22% w/v for collagen hydrolysate, rabbit collagen glue, and keratin hydrolysate, respectively) by electrospinning. The objectives of this study were (1) to extract and characterize the collagen hydrolysate (HC10), collagen glue (RCG), and keratin hydrolysate (KH) from bovine tanned leather by-products, pickled rabbit skin, and sheepskin wool, respectively; (2) to characterize the protein-based nanofibers obtained by electrospinning for potential biomedical field application; and (3) to demonstrate that the structure of protein-based nanofibers still preserves the secondary collagen structure without being totally affected by the extraction and electrospinning processing conditions. The nanofibers’ surface and mechanical properties have been confirmed using scanning electron microscopy coupled with the energy-dispersive spectroscopy (SEM-EDS) technique, attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR), differential scanning calorimetry (DSC), and indentation tests (Nanoindenter XP). The biocompatibility (in vitro cytotoxicity) of protein-based nanofibers prepared by electrospinning was also evaluated highlighting the perspective of using protein-based nanofibers in tissue engineering.The novelty of this research includes the production of nanofibers from low-molecular weight collagen and keratin hydrolysates with a high content of proteins  with a melting point of 65 °C and a density of 1.4539 g/cm3 was used. PEO shows good biocompatibility and low toxicity and it is soluble in toluene, tetrahydrofuran, hot water, dimethylformamide, and chloroform, which are solvents generally used in the electrospinning process of biomaterials [Chitosan from crab shells, which is highly viscous (Sigma) in the form of crystals, is characterized by structural viscosity 1267 MPaxs and sulfated ash 0.2%. Poly(ethylene oxide) (PEO), Materials . Glacialw/w), at 60 °C by using the Hei-VAP Rotary Evaporator , at which the collagen hydrolysate viscosity increased enough for developing spinnable properties. Intermediary processes for total separation of chromium traces were done by successive filtrations through 0.45 μm pore size media and decantation after alkaline and enzymatic hydrolysis stages [Collagen (HC10) was extracted from bovine tanned leather by-products by chemical enzymatic hydrolysis in alkaline condition using calcium hydroxide  at 80 °C and Alcalase 2.4 L (Novozymes) at 60 °C . HC10CC s stages . The rabs stages . The yiew/v) chitosan solution was obtained by dissolving of chitosan in 65% (v/v) aqueous acetic acid under magnetic stirring at 1000 rpm, and 60 °C for 4 h until a clear solution was obtained. HC10CC-chitosan solution with 13.6% (w/v) HC10CC was prepared by mixing HC10CC and chitosan solutions. RCG-acetic acid solution was prepared by mixing RCG with 40% (v/v) acetic acid solution, as the amount of RCG reached 25% (w/v). KH was gently mixed with 10% (w/v) aqueous PEO solution, as the amount of KH reached 22% (w/v). All prepared solutions were sonicated for 30 min to obtain homogeneous solutions.Concentrated collagen hydrolysate solution was prepared by dilution in distilled water at a weight ratio of 1:1. Previously, 1.5% , which consists of a syringe pump, a high-voltage power supplier, and a grounded conductive drum collector. Each protein/polymer/solvent solution was loaded into a Teflon syringe (10 mL volume) and fed through the tubing with a metal 21 gauge needle attached at the end. The electrospun HC10CC-, RCG-, and KH-based nanofibers were collected on a drum covered with aluminum foil. The optimal parameters for electrospinning process are summarized in w) were evaluated by validated in-house methods [2NO3 and 10% MeOH of 1 mL min−1, injection volume of the sample of 100 μL, and temperature of 35 °C for detectors and column. The GPC system was calibrated using polyethylene glycol standards with molecular weights in the range of 1000 to 16,000 g/mol.Electrical conductivity was measured with the C1010 Conductometer , with 1–100 mS/cm scale, according to EN 27888, and the viscosity was determined using a DV2T™ Viscometer . Polydispersity and zeta potential characteristics were investigated using a Zetasizer Nano-ZS device . Total nitrogen and protein substances, dry substances, and total ash were measured according to ISO 5397, EN ISO 4684, and EN ISO 4047, respectively. Aminic nitrogen and average molecular weight (M methods . Gel perw and number average molecular weight (Mn) were performed with the Agilent GPC/SEC Software . Circular dichroism spectra were recorded on a Jasco J-815 Spectrometer , and the results were processed with Dichroweb software. HC10CC, RCG, and KH extracts were cast and the films obtained were analyzed by ATR-FTIR in comparison with the electrospun protein-based nanofibers.Calculations of the MAll measurements were carried out at room temperature (25 ± 2 °C) in triplicate for each sample.The morphology and diameter of type of each electrospun protein-based nanofiber were observed by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS), using a Quanta 450 FEG scanning electron microscope  equipped with a field emission gun at a 1.2 nm resolution. The specimens were gold-sputtered prior to imaging, in order to increase their electrical conductivity. The diameters of minimum 50 nanofibers were measured and the results were processed using Origin 2016 software .−1 resolution in a 4000 to 750 cm−1 range, using air as background.Attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) analysis was done using an INTERSPEC 200-X Spectrophotometer , in transmittance mode. All spectra carried out both on the prepared films and electrospun nanofibers represent the average of 3 scans recorded at 2 cme, Calorimeter . Samples were heated from ambient temperature up to 250 °C at a heating rate of 10 °C/min. DSC curves were processed with the aid of STARe 9.10 software from Mettler-Toledo .DSC measurements of obtained protein-based nanofibers were determined using a Mettler Toledo (Model DSC 823E’) and damping or viscous component  [Eʺ/E’.The viscoelastic properties of specimens were measured by dynamic nanoindentation with a Nanoindenter XP , using the dynamic testing Continuous Stiffness Measurement (CSM) module. For small-scale samples this technique can be used to locally attain the same information as dynamic mechanical analysis (DMA) in the case of bulk samples ,38. The lus, Eʺ) ,40. The 2. Cytotoxicity of the samples was evaluated by the indirect contact method according to ISO 10993-5 standard. Each of the electrospun nanofibers was immerged in MEM supplemented with 10% FBS at 37 °C for 24 h to obtain the extraction medium. Fresh culture medium was used as negative control. L929 cells were seeded (5 × 104 cells/mL) in 96-well tissue culture plates and, after 24 h, the culture medium was replaced with varying concentrations of the extraction medium . Cells were maintained in standard conditions for 24 and 72 h when quantitative (MTT and LDH assays) and qualitative (cell morphology) analyses were performed.L929 mouse fibroblasts (European Collection of Authenticated Cell Cultures—ECACC) were chosen as a cell model to assess the cytotoxicity of the electrospun HC10CC, KH, and RCG nanofibers. Cells were grown in Minimum Essential Medium  supplemented with 10% fetal bovine serum  and 1% antibiotics  at 37 °C in a humidified atmosphere with 5% COTo evaluate the cell viability, cells were incubated with 0.25 mg/mL 3--2,5-diphenyltetrazolium bromide (MTT) solution for 3 h at 37 °C, as previously described by Craciunescu et al. . The insThe cytotoxicity of the electrospun nanofibers was evaluated by measuring the amount of lactate dehydrogenase (LDH) released into the culture medium when cells are damaged or under stress . At 24 aGiemsa staining was used to assess cell morphology after 72 h of cell incubation in the presence of the samples. Images of the L929 cells were acquired using an Axio Observer D1 inverted microscope and AxioVision 4.6 software .p < 0.05 were considered statistically significant.All values were expressed as a mean value ± standard deviation (SD) of three independent samples (n = 3). Statistical analysis of the biocompatibility data was performed using analysis of variance (ANOVA) (95% significant level) on each pair of interest and differences at The main physical-chemical characteristics of collagen hydrolysate (HC10) before and after concentration (HC10CC), and RCG and KH extracts are shown in w were determined according to the validated in-house methods and compared with the results obtained by GPC in Collagen hydrolysate (HC10) is an accessible and inexpensive resource of collagen, which was not used in typical electrospinning processes due to its low molecular weight (3.95 kDa) and low viscosity (1.5 cP) in water . Concentw protein extract by GPC are in good agreement with those determined using the aminic nitrogen method  spectroscopy was performed .It was found that after the extraction processes, the collagen and keratin molecules were not totally denatured; α-helix, β-strand (the unity of β-sheets), and β-turns were identified as the most important secondary structures . It is pThe energy-dispersive X-ray (EDS) patterns of nanofibers obtained by electrospinning and their elemental composition are shown in The energy-dispersive X-ray (EDS) patterns of electrospun proteins nanofibers exhibit elements of the peptide functional groups of the collagen , such as carbon, oxygen, and nitrogen in the ranges 50.79 to 58.63 wt.%, 21.77 to 28.68 wt.%, and 8.77 to 21.36 wt.%, respectively. The nitrogen peak increased in the case of RCG nanofibers. According to the EDS analysis of electrospun RCG and KH nanofibers, they contain a small amount of sulfur . Keratin contains cysteine amino acids that form inter- and intramolecular disulfide bonds, responsible for a high stability of this protein [The purpose of ATR-FTIR spectroscopy is to demonstrate that the partial triple helix structure of protein films is preserved both for protein extracts and electrospun protein-based nanofibers. −1 and 2935 cm−1 assigned to N–H stretch coupled with hydrogen bond (Amide A) and asymmetrical stretching of –CH2– (Amide B), respectively, see −1, 1532 cm−1, and 1246 cm−1 corresponding to the C=O stretch (hydrogen bond coupled with CN stretch (Amide I)), N–H bend coupled with C–N stretch (Amide II) and C–H stretch (Amide III), respectively [−1, 1407 cm−1, 1326 cm−1, 1246 cm−1, 1160 cm−1, and 1027 cm−1 were observed at all proteins before and after electrospinning, which were assigned to the –CH2– bend, COO– symmetrical stretch, CH2 wagging vibration, COOH group, and C=O stretch [−1, corresponding to the stretching of the CH2 group arising from the addition of PEO [−1 (–CH2–)  in air during electrospinning process [2– (1454 cm−1) [< 100 cm−1 [The assignment for individual peaks of HC10CC extract film indicates the presence of the prominent groups around 3264 cmectively ,44,45, an of PEO  and 2915 (–CH2–) c. In thetructure  was move process . The eff100 cm−1   test. Viscoelastic properties of electrospun HC10CC nanofibers could not be measured due to the difficulty to prepare proper specimens. From Another objective of this study was to evaluate the biocompatibility of HC10CC, KH, and RCG nanofibers using the MTT assay, which evaluates the activity of mitochondrial dehydrogenases, and the LDH assay, which investigates cell membrane integrity by quantifying the LDH enzyme released in the culture medium upon cell lysis. The MTT results showed that all tested protein-based nanofibers had no cell cytotoxic activity on a wide range of concentrations. The percentages of cell viability were higher than 80% (non-cytotoxic effect) within the concentration range of 0.1 to 1 mg/mL for all the tested samples at both exposure times (24 and 72 h) .p < 0.05). Similar cell viability values were obtained after 24 h for RCG sample at the concentrations of 2.5 and 5 mg/mL , whereas a moderate cytotoxic effect was observed after 72 h (71.58% at the concentration of 2.5 mg/mL and 70.91% at the concentration of 5 mg/mL).HC10CC nanofibers were also cytocompatible at the concentration of 2.5 mg/mL, whereas a moderate cytotoxicity was recorded at the concentration of 5 mg/mL after 72 h (cell viability of 73.43%). Cell viability was maintained above 80% for KH nanofibers at the concentration of 2.5 mg/mL, but significantly decreased below 50% at 72 h post-seeding , except for KH sample at a concentration of 5 mg/mL, and also after 72 h of culture, within the concentration range of 0.1 to 1 mg/mL .These results suggest that none of these nanofibers exhibited cytotoxic effects at the above mentioned concentrations. However, higher levels of LDH activity were recorded after 72 h for HC10CC at the concentration of 5 mg/mL and for KH and RCG nanofibers at the concentrations ranging between 2.5 and 5 mg/mL . These rThe quantitative results of cell cytotoxicity were confirmed by cell morphological analysis after Giemsa staining. Light microscope images revealed that L929 fibroblast cells exposed to different electrospun nanofibers maintained their normal morphological appearance, similar to that of the control j, up to w/v) chitosan dissolved in 65% aqueous acetic acid, as the amount of HC10CC reached 13.6% (w/v). In this case, the voltage was decreased to 18.81 kV. Chitosan is a unique cationic polysaccharide, an abundant and renewable natural polymer that shows many intrinsic properties, such as antioxidant, lipid-lowering and antimicrobial activities, film-forming and gelling properties, encapsulation potential, biodegradability, biocompatibility, non-toxicity, antitumor, antimicrobial, and adsorption activity [The electrospinning process represents a sustainable nanotechnology addressing large-scale material production. In this paper, nanofibers of indigenous natural resources based on collagen from bovine hides, rabbit skin glue, and sheep wool keratin originated from valuable by-products from fur and leather industry were successfully obtained by electrospinning. In order to set the optimal parameters for achieving the electrospun protein-based nanofibers, different polymers and solvent concentrations, amount of protein, flow rate, distance from the needle to collector, and voltage were tested. Concentrated collagen hydrolysate (HC10CC) shows a higher viscosity as compared to diluted collagen dispersion and associative properties, which make this material suitable for electrospinning in combination with the chitosan solution. Good spinnable properties of collagen hydrolysate are obtained by the increased viscosity with its concentration . Electriactivity ,52,53. Eactivity ,55, whicactivity . The spiactivity ,54. The activity . KH dissactivity . These sactivity . By addiws of protein extracts evaluated and validated by in-house methods are in good agreement with the GPC determinations. The structure of protein extracts evaluated by circular dichroism spectroscopy showed that the secondary structure of proteins was partially preserved after extraction conditions. The helix structure in the case of KH after alkaline extraction was also reported by other authors [The M authors .2- with value of about 1. The spectra of protein extract films revealed the wavenumber difference between Amides I and II < 100 cm-1 compared with those of electrospun protein nanofibers. This spectral behavior suggests that the protein chains were reorganized during electrospinning process, as ß-sheet and ɑ-helix conformation secondary structures. Electrospun RCG nanofibers showed a wavenumber difference ˃100, which indicates the presence of denatured collagen. The partial helix identification in electrospun protein-based nanofiber structures as compared to the protein extract films is a new discovery of this research with great potential for advanced biocompatible material design as compared to similar known products based on biopolymers. The triple helix structure of electrospun HC10CC nanofibers has been confirmed by other authors [The SEM observations of electrospun protein-based nanofibers revealed that the HC10CC resulted in larger nanofiber size (171.9 nm) than RCG 89.93 nm) and KH nanofibers (142.2 nm). The large nanofibers size of HC10CC could be due to the molecular weight of collagen hydrolysate of 17.8 kDa. Fibers with a diameter larger than 2 μm were obtained in the case of drug-loaded collagen/PLA , due to 9.93 nm a authors , althoug authors .DSC analysis reveals that the electrospun HC10CC, RCG, and KH nanofibers showed an improvement in their thermal stability, as compared with the denaturation temperature of native hydrated collagen (65 °C) . This faThe E′′ and loss factor of the electrospun RCG nanofibers show a continuous increase over the range of frequency used , while the storage modulus stays constant up to 20 Hz, showing a slight decrease at 45 Hz. All measured mechanical parameters for electrospun KH nanofibers are fairly constant over the frequency range; both storage and loss moduli are several tens of times larger than for electrospun RCG nanofibers, while the loss factor is 5 to 10× larger over on the tested frequency range. Regarding the energy dissipation capacity, this material proves superior capabilities compared to RCG nanofibers. The results can be explained by the smaller diameter size of RCG nanofibers compared to KH nanofibers. It is possible that the hard substrate of electrospun KH nanofibers influences the deformation of nanofibers.These results, which combine both in vitro quantitative and qualitative methods, show good cytocompatibility of the electrospun KH and RCG nanofibers at concentrations ranging between 0.1 and 1 mg/mL, whereas HC10CC nanofibers exhibit no cytotoxic activity up to a concentration of 2.5 mg/mL. Our observation is consistent with previous studies, which reported biocompatible electrospun keratin and collagen/chitosan matrices that are suitable for tissue engineering and wound healing ,63,64. T2– and the wavenumbers difference between Amides I and Amides II. A partially preserved native structure of HC10CC and KH nanofibers was confirmed by SEM analysis, while the DSC analysis showed an increase in the denaturation temperature as compared with native collagen. KH nanofibers showed superior dynamic mechanical properties as compared to RCG nanofibers. Finally, in vitro experiments demonstrated the cytocompatibility of all electrospun nanofibers, with HC10CC presenting the most promising result, which recommends the possible use of keratin nanofibers for the bone tissue engineering, while the collagen and rabbit collagen glue nanofibers can be used as biomimetic extracellular matrix for the regeneration of tissues.Different proteins from bioactive by-product resources were extracted and characterized in order to obtain electrospun nanofibers. The experiments showed that the concentrated collagen hydrolysate extracted from bovine leather by-products, rabbit skin collagen glue, and keratin hydrolysate from wool waste have the properties required for electrospinning processing. The concentrated collagen hydrolysate (HC10CC)–chitosan, RCG–acetic acid, and KH–poly(ethylene oxide) (PEO) solutions were straightforwardly processed by electrospinning. Accordingly to the circular dichroism spectroscopy performed on the extracted protein, it was found that the extraction process did not totally denature collagen structure. Preservation of the helical structure of collagen was also assessed by measuring the ratio between Amide III to the absorption band of –CH