Although there are many machining studies of carbon and glass fiber reinforced plastics, delamination and tool wear of basalt fiber reinforced plastics (BFRP) in edge trimming has not yet studied. This paper presents an end milling study of BFRP fabricated by resin transfer molding (RTM), to evaluate delamination types at the top layer of the machined edge with different cutting conditions  and fiber volume fraction (40% and 60%). This work quantifies delamination types, using a parameter Sd/L, that evaluates the delamination area (Sd) and the length (L), taking into account tool position in the yarn and movement of yarns during RTM process, which show the random nature of delamination. Delamination was present in all materials with 60% of fiber volume. High values of tool wear did not permit to machine the material due to an excessive delamination. Type II delamination was the most usual delamination type and depth of cut has influence on this type of delamination. Nowadays, aeronautical and automotive manufacturers use fiber reinforced composites, because of their properties of light weight and high strength . In manyMachining damage is unavoidable due to the high mechanical resistance of the fibers, which causes excessive tool wear and poor surface finish . In edgeStudies on delamination are focused on drilling and milling (slot milling and edge trimming). Main factors that affect delamination are the tool wear, determined by the machining conditions, tool geometry, and volume and orientation of fiber in the composite material ,7.d = Dmax/Do) is calculated as the ratio of the maximum diameter of the delamination zone (Dmax) to the drill diameter (Do). Secondly, the adjusted delamination factor (Fd) represents the conventional delamination factor (Fd) plus the damage area contribution. Some authors have used this delamination factor to evaluate spindle speed, feed rate and the point angle in delamination of carbon fiber reinforced plastics (CFRP) with response surface methodology [In drilling, a study based on digital image analysis defines different factors to evaluate delamination . Firstlyhodology  generatid) and surface roughness using neural network and genetic algorithm in glass fiber reinforced plastics (GFRP). As conclusion, Fd increases with feed rate and decreases with spindle speed [In milling works, there are mainly two different processes studied: slot milling and edge trimming. In slot milling, recent studies evaluate the occurrence and propagation of delamination in CFRP  concludile speed .In edge trimming, surface delamination is random . Damage Most studies that approach fiber reinforced composites machining are focalized in glass and carbon fibers, however, there are almost no studies with basalt fiber reinforced plastic (BFRP) . Basalt In this paper, a plain weave basalt fiber reinforced plastic is milling with edge trimming in order to study delamination. Cutting conditions  and fiber volume fraction are varying, as tool wear is evaluated. Tool selection takes into account a commercial tool suitable for edge trimming, with a high productivity due to a long tool life for chosen cutting parameters and with a high cutting length. This work studies and quantifies delamination types depending on the position that the tool hits the yarn, and stablishes parameters to compare different experiments.Basalt fiber reinforce plastic (BFRP) laminates used in this study were manufactured using RTM process. The material was a rectangular (420 mm × 260 mm) bidirectional panel with plies of plain weave basalt fibers impregnated with an epoxy resin . Fiber orientation was 90°. Edge trimming operation (down milling) was conducted using a Kondia B-500 milling machine , with a spindle power of 6 KW and a maximum rotation speed of 6000 rpm. A Mitsubishi Materials milling tool holder of diameter 25 mm , with two exchangeable uncoated carbide cutting inserts  was used for machining.Clamps were used in the milling machine to avoid vibrations and displacement of the laminate, leaving the minimum cantilevered possible. A film bag involving machining zone was used to avoid dust .Cutting conditions  and material characteristics (fiber volume fraction) were considered to study delamination, taking into account the flank wear of the tool. The Taguchi’s method for four variables at two-levels was used to elaborate the design of experiments. 8 orthogonal array was selected for determining combinations of factor levels to use for each experimental case. 8 orthogonal array. Due to the time consumed for each experiment, a full factorial design was not used, in order to reduce the number of experiments. No replicas were done, because of the random nature of delamination.In this paper, a LIn every experiment, a tool with new inserts machined without coolant for 80 min. Different stopping times , or cutting times (Tc), were made to measure delamination and flank wear (Vb) in both cutting inserts. Measuring of delamination and flank wear are carried out recording and analyzing images of the laminate material and the clearance face of the tool, respectively. An Olympus SZ61 microscope  and image processing software  with pixel calibration was used to measure delamination and flank wear. In flank wear, measurements were repeated two times in every insert and a maximum value was chosen for every experiment.In the experimental results obtained in this study, laminates with a 40% of fiber in volume do not present delamination a, becausDelamination can be classified in three Types  , as showFor each delamination type, the maximum value (Vmax) and the surface occupied (Sd) were measured . Only VmIn Experiment 2, cutting conditions were the worst  and flank wear increased very highly and no cutting was possible after 30 min. This experiment only shows values at 5, 10 and 30 min. High tool wear depends on high cutting speed and high feed per tooth .In Type I delamination, Vmax value is less than yarn size, and the highest values are with large feed per tooth. In Type II delamination, in most cases, Vmax values are larger than depth of cut (ap). This can be explained because, originally, the fill yarn has a sine wave form and after cutting the yarn is stretched , accordiWhen ap is less than yarn size, Type I delamination can be larger than Type II.In Experiment 3 at 80 min and in Experiment 4 at 30 min, values of Vmax were very high because Type II and III delamination were superimposed. In these experiments, Types I and III were lower than Type II, being Type II the most critical delamination.Delamination measurement is needed to justify the phenomenon that occurs when machining a plain weave sample. Relation between fiber orientation and position in the laminate to machining direction is an important parameter. Position fiber refers to the point where tool hits the width of the yarn. Two different kinds of delamination can be found: uniform and sine wave form. On the trimmed edge, delamination is uniform if the machining direction is aligned with fiber orientation. Delamination can be low or high, depending on the position the tool hits the yarn . Low delSine wave form is appreciated when machining direction is slightly inclined (α°) in respect to the fiber orientation , due to To compare delamination types among experiments a delamination parameter (Sd/L) is needed. In this parameter, a length (L) of 80 mm is evaluated, as well as the measured area of each type of delamination (as shown in Xd measurement is difficult. It can only be measured in the edges of the laminate (left Xd and right Xd). These values allow the calculation of the machining path of the tool on the yarn. Theoretical values of Xd did not coincide with observed values in every fill yarn along the length of the laminate, because plain weave form fibers move and deform in the mold during the RTM process because of injection pressure and plain weave manipulation. Due to this, expected area (Sd) value in Type II delamination did not coincide with the measured value.A qualitative trend can be observed, distinguished between, if the cutting tool path was inclined or not. If machining cutting (edge trimmed) advances to a yarn crossing, Sd decreases, and a value lower than expected is obtained (Sd ↓). If machining cutting path advances to a yarn center Sd increases, and a value higher than expected is obtained (Sd ↑). In every experiment, it can be explained why Sd is different than expected .If the yarn direction and cutting path are coincident, Sd values in Type II of delamination are uniform  along thIn every experiment, parameter Sd/L is represented by each delamination type . Values Results of Sd/L, taking into account values of Xd, are shown in High cutting conditions provokes high delamination and, in consequence, high tool wear, avoiding machining. In In Experiments 1, 3 and 4, after machining 80 min, flank wear does not reach the maximum tool wear to avoid machining, having a linear behavior and maintaining a low value (maximum flank wear tool is 0.073 mm), allowing machining until 240 m. For this reason, delamination is low and very similar in all experiments .This paper evaluates delamination in edge trimming of basalt fiber reinforced plastics (BFRP), manufactured by resin transfer molding (RTM), varying cutting conditions  and fiber volume fraction. Cutting tool selection has been an important issue, allowing machining large cutting lengths and maintaining a low flank wear.Delamination appears in the top layer of the laminate with a high fiber volume fraction (60%). If fiber volume is lower, resin layer at the top prevent delamination defect. In plain weave sample machining, relation between fiber orientation and position in the laminate to machining direction generate two different kinds of delamination: uniform and sine wave form.Three types of delamination  have been evaluated, increasing their values as cutting speed and feed rate increases. Delamination values are maintaining in low values, allowing cutting lengths until 240 m, except with high cutting conditions. In this case, cutting is not possible for a high tool wear, which provokes high delamination.Delamination prediction is not an easy task, because of its random nature. This is due to the movement of fibers during the RTM process and the position of the trimmed edge to the yarns. To explain delamination values of each type, a parameter relating surface areas and length (Sd/L) has been evaluated, together with the distance of the warp yarn from the trimmed edge until the next dip below the crossing fill yarn (Xd).Type II delamination is the most usual. In Type II delamination the maximum value of fiber protrusion is higher than depth of cut, because originally the fill yarn has a sine wave form and after cutting the yarn is stretched. For this reason, depth of cut is an important variable in delamination values.


Mentorship plays a critical role in the success of academic radiologists. Faculty members with mentors have better career opportunities, publish more papers, receive more research grants, and have greater overall career satisfaction. However, with the increasing focus on clinical productivity, pressure on turn-around times, and the difficult funding climate, effective mentoring in academic radiology can be challenging. The high prevalence of “burnout” among radiologists makes mentorship even more important. This article reviews benefits and challenges of mentorship in academic radiology, discusses how to institute a faculty mentoring program, examines different types of mentoring, and reviews challenges related to diversity and inclusion. Mentorship plays a critical role in the success of academic radiologistsMounting pressures on academic radiologists in a changing healthcare environment can lead to physician burnoutMentoring is an effective means to achieve career satisfaction and help prevent burnoutEstablishing a mentoring program in academic radiology requires the identification and training of effective mentors, and mentees benefit from multiple mentors for different needsWomen and underrepresented minorities in medicine often do not have access to mentors but benefit most from a mentoring relationshipMentorship plays a critical role in the success of academic radiologists. A mentor helps a mentee reach both personal and career goals by functioning as an adviser and role model while providing support and sharing knowledge . MultiplWhile most radiology residency programs have incorporated formal mentoring programs for residents , 12–14, This article reviews the benefits and challenges of mentorship in academic radiology, discusses how to institute a faculty mentoring program, examines different types of mentoring, and reviews challenges related to diversity and inclusion.A career in academic radiology requires balancing competing demands from clinical work, education, and research. Reasons for junior faculty to embark on a career in academic radiology include opportunities for teaching, research, and having an interesting mix of clinical cases, while deterrents include insufficient financial compensation, lack of academic time, high clinical workload, and the long time for promotion . Most faAcademic radiology has experienced major changes over the past decades , with inJunior and new faculty members typically focus on managing the clinical workload, trying to “survive” in a new hospital system with new colleagues and often growing family responsibilities , with liWhile most academic radiologists who do research are “clinician scholars” who are not dependent on external funding, a small percentage of academic radiologists are able to obtain government or industry-funded grants . Such fuOver the last decade, there has been an increasing focus on professionalism in academic medicine. Teaching and assessing competence in professionalism is now an explicit expectation for Accreditation Council Graduate Medical Education (ACGME) accredited programs in radiology ; howeverGenerational differences, increased diversity in the workforce, and the use of social media can also add to misunderstandings of professional conduct . MoreoveThe current healthcare climate poses several challenges to academic radiologists. Successful mentoring is more important than ever for retaining and managing the current and upcoming demands of junior faculty, and to ensure the future of radiology .Several studies have examined key components of a successful mentoring program in academic radiology , 15, 43.Setting clear expectations for mentors and mentees, reviewing a mentorship agreement which states the ground rules of the mentorship relationship, and creating a career development plan with short- and long-term goals, which can be revisited during the mentoring meetings, are key components of our mentoring program. Additional goals of the program are academic promotion and preparing mentees for the annual career conference with their division heads. In this regard, mentors and mentees receive dedicated lectures and written material on promotion criteria and how to make the best of their annual career conference.Regular meetings among the mentees of our program help to exchange ideas and allows for peer-mentoring. We also provide funds to help junior faculty members to hire medical students or college students to help with research and scholarly projects. These interactions give the mentees the opportunity to become mentors themselves for college or medical students.Finally, assessment of the overall effectiveness of a mentoring program can be obtained through evaluations on career satisfaction, attrition rates of junior faculty, promotion, research-related metrics, such as the number of scientific publications, scientific talks, and grant submissions, teaching-related metrics, such as the number of courses taught and educational publications, and clinical-related metrics, including the development of new clinical care and participation in clinical committees. Feedback from these evaluations can help radiology departments to understand the value of mentorship and allows them to tailor their resources to provide effective support to junior faculty.The identification and preparation of effective mentors are key to the establishment of a successful mentoring program , 43. SevThe main barrier to becoming a mentor is lack of time , 14, 16,Malignant mentor behaviors have been described by Chopra et al., which include taking the mentees’ ideas and labeling them as their own, exploiting the mentee by giving them low-yield activities that are not advancing their careers, or dominating the mentee, not allowing input from others, which can lead to isolation of the mentee . Those bMentees play a critical role in the success of the mentor-mentee relationship, and mentees need to be instructed about what is expected from them. This can be accomplished through seminars and the distribution of written material prior to entering into a formal mentoring relationship. Studies have indicated that mentees should be proactive and should take initiative for the mentoring relationship , 50. TheMentee missteps often stem from uncertainty about their responsibilities and the mentors’ failure to address them. Vaughn et al. described common mentee missteps and solutions on how to overcome them . AwareneIdentifying helpful mentors for junior and newly hired faculty is key to a successful transition to a new faculty position . HoweverSeveral ways to match mentors and mentees have been described, ranging from self-selected to assigned mentors . The impIn our radiology mentoring program at the MGH, we pair junior faculty members with mentors outside of their division. Mentees often already have mentors within their division and having mentors in the same division can lead to conflicts of interest regarding shared or needed resources or when conflicts arise within the division. Pairing mentors and mentees across divisions also fosters better communication within the department. Pairing of mentors and mentees occurs with input from the mentor and mentee with guidance from the director of the mentoring program. The director of the mentoring program meets with each mentee and suggests potential mentors based on the right chemistry and needs. Both, mentors and mentees, have input in the final pairing.Some programs prefer a more natural matching process by which mentees independently choose their own mentors , 15. MenWhile mentorship is often viewed as a dyad with a senior mentor and a junior mentee, peer-mentoring, which encourages junior faculty members of similar rank to collaborate and exchange ideas, is an important part of a mentoring program. Because of the inherent equality of the participants who are often at similar stages in their personal life, participants are often more comfortable discussing professional and non-professional topics, such as the balance between work and family . MoreoveSpeed mentoring is an organized event in which a junior faculty member is paired with six senior faculty members for a series of 10-min encounters . A speedWomen are underrepresented in the radiology workforce and women only occupy a minority of leadership positions without a significant change over the last decade , 63. TheFemale faculty members are less likely to advance academically than their male colleagues of comparable seniority . DemandsMentoring is particularly important for the success and retention of faculty underrepresented in medicine (URiM) , 72 who Radiology departments should identify a diverse group of mentors who are able to meet the needs of a diverse faculty. Providing a network of diverse mentors is key to a successful and inclusive mentoring program that is able to provide equal opportunities and advancement to all faculty.Mentorship in academic radiology is more important than ever given the mounting pressures of a changing healthcare environment. Mentorship for junior faculty promotes job satisfaction and career advancement, helps avoid burnout, and increases faculty retention. While mentorship is traditionally viewed as a senior mentor and a junior mentee, diverse mentorship teams and peer-mentoring are important components of a successful mentoring program. Academic institutions should recognize the value of mentoring and ensure equal opportunities and advancement of a diverse faculty.


OBJECTIVES/SPECIFIC AIMS: Treatment of acute myeloid leukemia (AML) is challenging, as apoptosis-resistant AML cells often persist within the bone marrow microenvironment despite chemotherapy. The overall goal of our laboratory is to identify and ultimately target the bone marrow factors that protect AML cells. METHODS/STUDY POPULATION: Using cell cultures, we previously reported that SDF-1 (CXCL12), an abundant bone marrow chemokine, induces apoptosis of isolated CXCR4+ AML cells, including freshly isolated bone marrow-derived AML cells from approximately one-third of AML patients. However, co-culture of AML cells with differentiating osteoblasts protected AML cells from apoptosis. RESULTS/ANTICIPATED RESULTS: Histone deacetylase inhibitors (HDACi) abrogated the ability of osteoblasts to protect AML cells and altered expression of matrix mineralization genes including tissue nonspecific alkaline phosphatase (TNAP). A different drug, cyclosporine A (CSA), similarly inhibited osteoblast-mediated protection of AML cells and reduced TNAP expression. Specifically targeting osteoblast TNAP via siRNA was sufficient to prevent osteoblasts from protecting AML cells in co-cultures. In addition, we are targeting TNAP enzymatically. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that targeting TNAP may be useful in AML treatment to render the bone marrow microenvironment more hostile to leukemic cell survival.


Although the precise neuropathological substrates of cognitive decline in Parkinson’s disease (PD) remain elusive, it has long been regarded that pathology in the CA2 hippocampal subfield is characteristic of Lewy body dementias, including dementia in PD (PDD). Early non-human primate tracer studies demonstrated connections from the nucleus of the vertical limb of the diagonal band of Broca  to the hippocampus. However, the relationship between Lewy pathology of the CA2 subfield and cholinergic fibres has not been explored. Therefore, in this study, we investigated the burden of pathology in the CA2 subsector of PD cases with varying degrees of cognitive impairment and correlated this with the extent of septohippocampal cholinergic deficit. Hippocampal sections from 67 PD, 34 PD with mild cognitive impairment and 96 PDD cases were immunostained for tau and alpha-synuclein, and the respective pathology burden was assessed semi-quantitatively. In a subset of cases, the degree of CA2 cholinergic depletion was quantified using confocal microscopy and correlated with cholinergic neuronal loss in Ch2. We found that only cases with dementia have a significantly greater Lewy pathology, whereas cholinergic fibre depletion was evident in cases with mild cognitive impairment and this was significantly correlated with loss of cholinergic neurons in Ch2. In addition, multiple antigen immunofluorescence demonstrated colocalisation between cholinergic fibres and alpha-synuclein but not tau pathology. Such specific Lewy pathology targeting the cholinergic system within the CA2 subfield may contribute to the unique memory retrieval deficit seen in patients with Lewy body disorders, as distinct from the memory storage deficit seen in Alzheimer’s disease. The clinical presentation of Parkinson’s disease (PD) displays a high degree of heterogeneity, not limited to the classical quartet of motor signs . It has Despite extensive clinicopathological studies conducted over the last few decades, the exact neuropathological substrate contributing to cognitive decline in PD remains unclear. The neuropathology of PDD is similar to dementia with Lewy bodies (DLB) , which iThere has been recent interest in the hippocampal CA2 subfield following better morphological and physiological characterisation of the hippocampal subregions , 62. It Based on evidence from existing studies, we hypothesise that cholinergic dysfunction contributes to progressive cognitive decline in PD and is associated with increased Lewy pathology within the CA2 hippocampal subfield. We aimed to study tau and αSN pathology burden in the CA2 subsector in PD cases with different degrees of cognitive impairment. We also investigated the degree of cholinergic degeneration in the CA2 subsector and the Ch2 to identify septohippocampal cholinergic pathway involvement. Finally, we explored associations between tau, αSN and cholinergic processes in the CA2 subfield.Post-mortem human brain samples used in this study were provided by the Parkinson’s UK Tissue Bank at Imperial College London  and in Scotland (SC037554)). Tissue sections containing the hippocampus and the nvlDBB were obtained.The diagnosis of PD was based on established clinical and neuropathological criteria , 43. In Only cases with available hippocampal sections with the CA2 region clearly visible, adequate tissue fixation and good tissue quality were selected. Cases were excluded if the clinical notes were incomplete or of poor quality. Hence, only cases with clinical follow-up within 24 months before death were included. Cases with extensive vascular lesions in the brain including cerebral infarcts, haemorrhage, severe CAA pathologies and small vessel disease, which may contribute to cognitive decline, were excluded. Cases with severe co-existing AD pathology (Braak tau staging IV or above) were also excluded. Finally, cases with co-existing neuropathology including tumours, demyelinating lesions, fronto-temporal lobar dementia, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease and other Parkinsonism pathologies including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and multiple systems atrophy (MSA), were also excluded.For the first part of the study, formalin-fixed, paraffin-embedded diagnostic slides (7 μm thick) containing the CA2 region of the hippocampus, stained for αSN and tau (Phospho-PHF-tau pSer202 + Thr205) were obtained from the tissue bank. Serial haematoxylin and eosin (H&E) stained slides were used as a guide for the identification of the CA2 region. We defined the CA2 region as the pyramidal cell layer parallel to the boundaries set by the ends of the granule cells of the external limbs of the dentate gyrus Fig. . This de2O2)/phosphate buffered saline  for 30 min at room temperature for quenching of endogenous peroxidase activity. Next, tissue sections were pretreated with autoclaving (25 min) in 0.01 M sodium citrate buffer (pH 6) before rinsing in PBS (3 × 5 min) and incubation with anti-ChAT antibodies  at 4 °C overnight. 2% rabbit serum  or 2% donkey serum  was added in the antibody diluent for blocking of non-specific staining.Cholinergic neurons in the nvlDBB (Ch2) and cholinergic varicosities in the hippocampal CA2 region were visualised using immunohistochemistry and immunofluorescence respectively for choline acetyltransferase (ChAT). For this study, tissue sections at a coronal level just anterior to the anterior commissure decussation were obtained for Ch2 quantification as the highest density of cholinergic neurons is found at this level  and signal amplification techniques were carried out. In the case of standard immunohistochemistry, sections were incubated with biotinylated rabbit-anti-goat secondary antibodies  for 1 h at room temperature. After rinsing with PBS (3 × 5 min), sections were incubated with avidin-biotin complex using the VECTASTAIN Elite ABC Kit  for 1 h at room temperature. Subsequently, tissues were washed in PBS (3 × 5 min) and visualised with 3′3-diaminobenzidine (DAB) (5 min). Tissue sections were then washed in distilled water (2 × 5 min) and briefly counterstained with haematoxylin. Finally, tissues were rehydrated and cleared through an increasing concentration of IMS/ethanol  and in xylene (2 × 5 min), before coverslipping with Distrene-Plasticiser-Xylene (DPX).For immunofluorescence, hippocampal sections were incubated with Alexa Fluor 488-conjugated donkey anti-goat secondary antibody  for 1 h at room temperature. Sections were then rinsed briefly in PBS (3 × 5 min) and incubated for 10 min in 0.3% Sudan black B dissolved in 70% ethanol to block endogenous autofluorescence by lipofuscin, before coverslipping and mounting with VECTASHEILD antifade mounting medium with DAPI .Double and triple immunofluorescence were used to investigate the relationship between αSN and/or tau pathology and cholinergic fibres in the hippocampus. Sections were dewaxed and rehydrated as described in the section above. For double staining with anti-ChAT and anti-AT8 tau antibodies, tissues were pretreated with autoclaving (25 min) in 0.01 M sodium citrate buffer (pH 6). Additional incubation in 80% formic acid (10 min) was needed for double staining with anti-ChAT and anti-αSN antibodies. Tissue sections were then incubated with various antibodies combinations overnight  at the Facility for Imaging by Light Microscopy (FILM) at Imperial College London. × 10 objectives , × 63 objective  with laser excitation at 405 nm (Diode), 488 nm (Argon multiline), 543 nm (HeNe) and 594 nm (HeNe) were used. Image capture and processing were performed using the Zen Black  software.Stained hippocampal sections were screened using a × 10 stage objectives to locate the CA2 hippocampal subfield and quantitative assessment of ChAT-immunopositive fibres was carried out under a × 63 stage objective, blinded to the clinical diagnosis, at the region of maximal ChAT immunoreactivity to ensure consistency of the assessment. The full thickness of the section was imaged with z-stacking and the maximal intensity projection was acquired post-acquisition using the Zen Black software. The image was exported and analysed using the ImagePro Plus  image analysis software as described in Fig. ChAT-immunostained sections were visualised and images captured with a light microscope (Olympus AHBT3 VANOX) with digital camera at × 4 stage objective. Basal forebrain cholinergic cell groups often consist of a compact and diffuse sector . For thip < 0.05.Statistical analysis was carried out and graphs were generated using Statistical Package for the Social Sciences  and GraphPad Prism software (Version 7.02). Data in this study was not normally distributed, hence Kruskal-Wallis and Mann-Whitney U tests were used for comparisons of ChAT-positive neurons and varicosities, and tau and Lewy pathology burden among the cases. Post-hoc pairwise analysis with Dunn’s correction was used for multiple comparisons. Spearman’s rho was used for correlation between ChAT-positive neuron count in the nvlDBB and ChAT-positive varicosities in the CA2 hippocampal region. Statistical significance is reached when In summary, 197 cases comprised of 67 PD, 34 PD-MCI and 96 PDD cases were selected for this study. All cases were matched in age at death, disease duration, and Braak αSN and tau stages Table . There wp = 0.0074) and PD cases without cognitive deficits (p < 0.0001)  or αSN  pathology.Semi-quantitative assessment of Lewy and tau pathologies were performed on diagnostic hippocampal sections. However, 7 diagnostic slides for tau analysis and 9 for αSN analysis had to be excluded due to poor tissue quality and the difficulty in accurately identifying the CA2 subfield. There was no difference in CA2 tau burden among PD cases with differing degrees of cognitive deficit, although a trend towards a higher burden in demented cases could be seen Fig. a. In terCases with tissue sections containing the nvlDBB at the coronal level just anterior to the anterior commissure decussation were obtained and immunostained for ChAT for the quantification of cholinergic neurons. Since the nvlDBB was not a routinely sampled area at the tissue bank, only 8 PD, 4 PD-MCI and 16 PDD cases with representative anterior basal forebrain section and suitable hippocampal sections were available for this part of the study Table . Cases wp = 0.0275) and PDD (p = 0.0207) cases compared with PD cases with no cognitive impairment  . No correlation between ChAT-positive varicosities in the CA2 and αSN burden  or tau burden  was found.Hippocampal sections of the selected cases were also immunostained for ChAT for the quantification of cholinergic varicosities within the CA2 subfield. A significant decrease in ChAT-positive varicosities was found in PD-MCI  were double or triple immunostained for ChAT, αSN and tau. A strong relationship between αSN and ChAT was found with double immunofluorescence Fig. . Nearly This study supports the hypothesis that pathology within the hippocampal CA2 subfield contributes to cognitive decline in pure PD cases with no or minimal co-existing Alzheimer’s pathology. In particular, a high burden of Lewy pathology differentiated cases with dementia from those without. In addition, the loss of CA2 cholinergic fibres appeared to be a more sensitive marker as it distinguished PD cases with cognitive impairment from cases with no reported cognitive deficit. The significant association of a high density of hippocampal CA2 Lewy pathology with dementia in PD was consistent with previous observations , 47. In The presence of hippocampal dopaminergic innervation has been extensively reported in rodents . HoweverRecent advancement in neuroscience techniques has led to better understanding of the precise function and connectivity of various hippocampal subfields. In particular, there has been a resurgence of interest in the CA2 region as its molecular signature was recently established in detail, distinguishing itself from the neighbouring CA1 and CA3 subfields , 24. SelIn neurodegenerative conditions, specific subfield predilections have also been seen in aggregated protein pathology . In AD, The connectivity of CA2 has been well described in rodents. Pyramidal neurons within the CA2 are the major output neurons to the CA1 and CA3 and they receive excitatory glutamatergic input from dentate gyrus and entorhinal cortex. They also receive input from other subcortical regions including the medial septum/ diagonal band, amygdala and hypothalamic nuclei. Thus, the CA2 was thought to be the integrating hub for cognitive and emotional functions . In fact


Toxoplasma gondii has received less attention. Our data showed that estradiol and progesterone induced parasitic cytosolic Ca2+ fluxes. This process required estrogen to enter the cytoplasm of T. gondii, and cGMP-dependent protein kinase G (PKG) and phosphoinositide-phospholipase C (PI-PLC) emerged as important factors controlling parasitic intracellular (IC) Ca2+ signals. Cytosolic Ca2+, which is regulated by estradiol, was mostly mobilized from acidic organelles. Moreover, cytosolic Ca2+ slightly increased MIC2 protein secretion and promoted the gliding motility and egress of parasites, thus enhancing the pathogenicity of T. gondii, as shown in our previous research. We subsequently determined that the main source of Ca2+ regulated by progesterone was a neutral store. In contrast to the findings of estradiol, progesterone reduced MIC2 protein secretion and inhibited the gliding motility of parasites, which may decrease their pathogenicity. Additionally, unlike in mammals, estradiol and progesterone had no effect on nitric oxide (NO) or reactive oxygen species (ROS) production in T. gondii.Research has shown that estrogen is present and plays a critical role in vertebrate reproduction and metabolism, but the influence of steroids on  Toxoplasma gondii is found worldwide, and this pathogen can infect a wide range of hosts, such as humans, livestock, pets, and wildlife. All karyocytes are considered to be definitive cells of T. gondii  of T. gondii for quite some time, the results have been inconclusive. Therefore, the study of estrogen-mediated rapid effects is a different approach for obtaining an in-depth understanding of this topic. Short-term actions, such as membrane-and/or cytosol-initiated mechanisms, involve a rapid response through the activation of signal transduction pathways mediated by membrane estrogen receptors (mERs) or G-protein-coupled receptors (GPCRs) and ion channels, modulating kinase activation and ionic fluxes, such as calcium fluxes  and long-term actions (hours to days), which depend on different receptors. There are currently no available reports concerning m fluxes . Becausem fluxes , the posT. gondii is accompanied by fluxes of cytosolic Ca2+. These fluxes are necessary for parasite motility and egress from host cells. During the T. gondii life cycle, the parasite invades host cells to create a parasitophorous vacuole (PV), where it divides and matures. Parasite development involves several steps: (i) gliding motility, (ii) conoid extrusion, (iii) secretion of specific proteins, (iv) attachment to the host cell, (v) active invasion, and (vi) egress. Previous studies have shown that intracellular (IC) Ca2+ fluxes are important for the initiation of gliding motility, microneme secretion, conoid extrusion, active parasite invasion, and egress (T. gondii and thus significantly contribute to the pathogenicity of T. gondii in mice (2+ induced by estradiol in these processes may be one factor in estrogen-promoted high pathogenicity.The life cycle of d egress . Our pre in mice . Therefo2+ indicators (GCaMPs) were used to develop a parasite strain for the observation of estrogen-induced Ca2+ signals in T. gondii. Our results showed that estradiol and progesterone induce the release of Ca2+ of T. gondii from different stores. Additionally, estrogen-induced Ca2+ fluxes relate to parasite gliding motility, microneme secretion, and egress.Genetically encoded CaT. gondii RH strain tachyzoites  and the RH-GCaMP6f strain  were maintained in vitro on Vero or HFF cells in DMEM  containing 25 mM glucose and 4 mM glutamine supplemented with 8% fetal bovine serum  and were incubated at 37°C with 5% CO2 in a humidified incubator. The medium was changed 12 h after inoculation. The RH-GCaMP6f strain was constructed and provided by Silvia N. J. Moreno, and the detailed plasmid information was reported previously (T. gondii were kindly provided by David Sibley at Washington University. The coding DNA sequence for GCaMP6f (Addgene) was amplified via PCR and cloned into a T. gondii vector for expression under the tubulin promoter.HFFs (human foreskin fibroblasts) and Vero cells (African green monkey kidney cells) were obtained from the Cell Bank of the Chinese Academy of Sciences . eviously . BrieflyT. gondii were measured using the probe 2′, 7′-dichlorofluorescein diacetate . Tachyzoites were pretreated with estrogen for different times and then harvested, after which they were incubated with 10 μM DCF-DA for 1 h at 37°C, washed twice with phosphate-buffered saline (PBS), and quantified utilizing flow cytometry. Estrogen-pretreated parasites using same method were used to measure NO activity in a chemiluminescence assay according to the manufacturer’s instructions .Intracellular reactive oxygen species (ROS) levels in 2, 142 mM NaCl, 25 mM HEPES, 5 mM KCl, 5.6 mM D-glucose, 1.8 mM CaCl2, pH 7.4). Estradiol , and progesterone  were added to the resuspension solution, and parasites were allowed to secrete for 15 min at 37°C. After centrifugation at 2500 rpm, the supernatant and pellet were collected for western blot analysis.Fresh tachyzoites were harvested, washed twice with PBS, and resuspended in extracellular (EC) buffer  were added to glass-bottom culture dishes and then heated to 37°C. Compounds were then added, and fluorescence images (3 min duration) were collected on an Olympus fluorescence microscope. The images were exported from Zen, and fluorescence intensities in the GFP channel were quantified in Image-Pro for each time slice.For time-resolved microscopy, purified RH-GCaMP6f parasites in IC buffer , and immunofluorescence images were acquired as previously described . For allt-tests using GraphPad Prism 5 . Statistical data are presented as the mean value ± standard error of the mean (SEM). P < 0.05 and P < 0.01 were considered statistically significant and very significant, respectively.Statistical significance between groups was evaluated by two-tailed unpaired Student’s 2+ release from parasitic IC stores, we used the genetically encoded Ca2+ biosensor GCaMP6s to monitor IC Ca2+ fluxes . We observed green fluorescence representing the flux of increased Ca2+ in response to estradiol  and progesterone . Our previous studies have shown that BSA-coupled estradiol cannot enter the cytoplasm of T. gondii , calmodulin, and M13 (a peptide sequence from myosin light chain kinase). When calcium is present, calmodulin undergoes a conformational change and binds M13 to generate green fluorescence. To determine whether estradiol and progesterone induce an increase in Ca. gondii . In FiguT. gondii exhibits several regular Ca2+ stores, such as the endoplasmic reticulum (ER), mitochondria, and some acidocalcisomes. Ionomycin has been reported to specifically mobilize neutral Ca2+ stores of T. gondii  treatment, indicating that Ca2+ had been mobilized from neutral stores. Subsequent treatment with progesterone no longer produced an obvious Ca2+ peak, meaning that most of the progesterone-mobilized Ca2+ had already been depleted by ionomycin. Therefore, progesterone-induced Ca2+ mostly originates from neutral stores. In contrast, treating parasites with ionomycin followed by estradiol still produced an obvious Ca2+ spike . These results mean that estradiol-induced Ca2+ does not originate from neutral stores, in contrast to progesterone.In general, . gondii . A peak 2+ stores are widely recognized as playing important roles in Ca2+ signaling. The addition of glycyl-L-phenylalanine-naphthylamide  has a swelling effect on the release of Ca2+, and cause Ca2+ to leak from lysosomal-like compartments . In contrast, progesterone still induced a large quantity of Ca2+ release, regardless of the treatment . Therefore, estradiol-induced Ca2+ is mostly derived from the acidic store of T. gondii, in contrast to progesterone.Acidic Caartments . We trea2+ influx into the mitochondrial matrix is mainly mediated by a Ruthenium compound-sensitive Ca2+ influx uniporter , indicating that increases of Ca2+ induced by the two estrogens did not originate from the mitochondrial matrix.Caniporter , which hlciparum . Rutheni2+ signals in malaria parasites  treatment, and a reduced Ca2+ spike was observed compared with the control group . These results showed that the estradiol and progesterone-induced Ca2+ spikes may be related to PKG.Previous studies have shown that cGMP-dependent PKG is essential to Caarasites . MBP146-hyzoites . To dete2+ spikes . These results indicate that PKG and PI-PLC are important factors in estrogen-induced calcium production. Of course, we cannot rule out the possible existence of other factors or signaling pathways that mediate this response, as estradiol and progesterone still induced a small amount of Ca2+ production under treatment with these inhibitors.U-73122  is a phosphoinositide-phospholipase C (PI-PLC)-specific inhibitor. Using the same method, we found that PI-PLC is required for the estrogen-induced Ca2+ fluxes and are essential for T. gondii motility . However, progesterone-induced MIC2 secretion  was inhibited. Additionally, we found that progesterone did not inhibit MIC2 expression (∼130 kDa), as there was no observed decrease in pellets compared with those in the DMSO group. Using a monoclonal MIC2 antibody, we found that the mature form of the MIC2 protein (∼100 kDa) was significantly decreased in progesterone-pretreated parasites compared with that in the estradiol and DMSO pretreatment groups . From the above experiments, we established that estrogen might modulate parasite invasion by modulating MIC2 protein secretion, as knocking out the MIC2 protein caused T. gondii to lose its invasion ability , and most T. gondii were in a motionless state . We then counted the percentage of parasites that could form complete loop motion trails. Under our experimental conditions, estradiol incubation increased parasite motility in a manner that was somewhat comparable to DMSO incubation . These findings indicated that estradiol treatment increased parasite motility, which likely depended on the alteration of Ca2+ fluxes in the parasites. To our surprise, progesterone inhibited the gliding of T. gondii and reduced MIC2 protein secretion.The + fluxes . We nextrescence . ParasitT. gondii affected parasite motility; hence, we reasoned that estrogen might promote active egress of T. gondii from host cells, which is triggered by changes in the ionic environment , whose egress rate was consistently observed to be 60% . In contrast to the results of estradiol treatment, the egress rate of progesterone-pretreated parasites was low, albeit still higher than that of the control group at 33% . However, we observed that most of the egressed parasites induced by progesterone did not migrate to neighboring cells due to low gliding motility . In addition, progesterone inhibited the maturation of the MIC2 protein, as shown in Figure 4, and we can speculate that progesterone may make it more difficult for egressed T. gondii to invade new cells for survival. In contrast, estradiol enhanced the vitality of T. gondii.We observed that estrogen treatment of extracellular ironment . When inT. gondii pretreated with progesterone and estradiol and found that the two estrogens enhanced the expression of PLP1 and promoted the egress of T. gondii .PLP1 (perforin-like protein 1) was found to be necessary for rapid egress, and PLP1-deficient parasites failed to egress rapidly from the PV after calcium ionophore treatment . TherefoT. gondii replication is the production of NO. In several in vitro studies, NO was shown to be involved in the killing of IC T. gondii .Estrogens have been reported to prevent IC peroxide accumulation, decrease ROS production , limit l. gondii . TherefoT. gondii invasion and proliferation, which has been discussed in our previous work, and Ca2+ signaling is crucial in parasite biology. Therefore, estrogen-induced Ca2+ fluxes may play an important role in the survival of T. gondii. In this study, we extend our understanding of the effects of estrogen on T. gondii by demonstrating that PKG and PI-PLC activities are important for the robust Ca2+ response elicited by estrogen. Furthermore, we determined the source of estrogen-induced Ca2+ to be distinct from different stores and used this phenomenon as the basis for explaining the relationship of estrogen with parasite gliding motility, microneme secretion, and egress.Estrogen is associated with 2+ is generated via modulation of the phosphoinositide-signaling pathway through binding to a surface receptor, most commonly a GPCR. However, in the present study, we found that BSA-coupled estradiol could not stimulate Ca2+ production. This result may indicate that GPCR-like membrane receptors did not mediate the observed Ca2+ release but that it occurred directly through estradiol entering the cytoplasm of T. gondii. Therefore, the pathway of estradiol-induced T. gondii Ca2+ fluxes remains to be further studied, and an estradiol receptor responsible for mediating IC calcium increases may reside in the cytoplasm of parasites.Previous studies have indicated that CaToxoplasma and Plasmodium spp.  generation.Previous reports have shown that the cyclic nucleotide second messenger cyclic GMP (cGMP) activates PKG and plays a vital role in the activation of apicomplexan egress and motility . Inhibitium spp. . Importa berghei  and eliclciparum . These cT. gondii microneme secretion. PA is a key factor in the microneme secretion-signaling pathway, and blocked production of PA inhibits microneme secretion, even when the release of Ca2+ is induced , and inhibition of PA production might be the mechanism through which progesterone reduced the secretion of MIC2. Microneme proteins are key factors in parasitic invasion, and knocking out MIC2 caused T. gondii to lose its invasion ability. Estradiol slightly increased the secretion of MIC2, which was one factor that enhanced the pathogenicity of T. gondii. However, progesterone inhibited MIC2 protein maturation and secretion, which may significantly decrease the parasitic invasion ability of T. gondii. Although progesterone promoted the egress of parasites from host cells, T. gondii may have been less pathogenic because of its reduced invasiveness. Actually our previous work showed that estradiol enhances the pathogenicity of T. gondii (Membrane phosphatidic acid (PA) level plays a critical role in  induced . In our . gondii , and proT. gondii. We speculate that progesterone may affect the function or expression of certain proteins to trigger egress, regardless of reduced motility. Moreover, egress is assisted by the release of TgPLP1, which forms pores in the PV membrane (PVM) and the host cell plasma membrane  have been found in this group . For examembrane . ProgestFigure 2C. Some reports (T. gondii at present. Because of this ambiguity, we cannot elaborate further.Estrogen receptors are found in mitochondria, and estrogens have substantial effects on mitochondrial biogenesis and metabolism, as well as on ROS generation . In our  reports  have ind2+ fluxes in T. gondii and further explains the relationship of estradiol and progesterone-induced Ca2+ with parasite invasion, gliding motility, and egress. Further work will be necessary to better understand the change in the pathogenicity of T. gondii during pregnancy.In summary, our research reveals estrogen-induced CaXZ, JL, and QL conceived and designed the experiments. XZ performed the experiments. XZ, HZ, and YF analyzed the data. QL contributed reagents, materials, and analysis tools. XZ and QL wrote the manuscript.The 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.


Fracture of a femoral component after modern unicompartmental knee arthroplasty is very rare. Although this is not the first case on this subject, no study has reported insufficient crimping as the cause of femoral component loosening that led to breakage of a metallic component. A 69-year-old man underwent medial unicompartmental knee arthroplasty for right medial knee osteoarthritis. His early postoperative course was good; however, the 1-year postoperative radiograph showed an apparent radiolucent line around the femoral component, and he occasionally had right knee pain. However, he had been followed up conservatively because he had been doing well even while doing heavy agricultural work. At 8 years after surgery, because breakage of the femoral component was found, revision surgery was performed using bicruciate-retaining total knee arthroplasty. The removed fractured femoral component revealed a thick cement mantle detached from the bone surface. The postoperative course of the patient after the revision surgery was excellent. We suggest that the causes of femoral component breakage include early implant loosening caused by uneven cement crimping of the femoral component to the bone and excessive loading stress as a result of heavy labour. Unicompartmental knee arthroplasty (UKA) was used for the first time in the 1970s for patients with single compartment osteoarthritis , 3. Howe2, respectively.A 69-year-old man presented with a 10-year history of right knee pain due to medial osteoarthritis. He worked as an agriculturist, and his weight and body mass index (BMI) were 70 kg and 27.6 kg/mHis range of motion of his right knee was 5°–135°, and the collateral and cruciate ligaments were intact. Preoperative radiography demonstrated the presence of medial osteoarthritis. Medial UKA  was performed because his symptoms did not improve with conservative treatment. Surgery was performed via the tibia-dependent cut technique using a spacer block, and cement-retained prostheses were implanted. A femoral component of size E, a tibial component of size 3, and an 11 mm polyethylene insert were placed. No complications were observed during the perioperative period. The postoperative knee range of motion was 0°–140°, and no knee instabilities were noted. No abnormalities and problems with the positioning of the component were noted on the postoperative radiograph .At the 1-year postoperative follow-up, the patient reported occasional right knee pain. Radiography revealed a radiolucent line around the femoral component . HoweverAt the 8-year postoperative follow-up, he complained of increasing right knee pain, and radiography revealed fracture of a metallic femoral component, loosening of the femoral component, and marked narrowing of the medial joint space . There wIntraoperatively, a fracture and loosening of the femoral component were observed Figures . There wInformed consent was obtained from the patient, and this case study was approved by the concerned institutional review board.Fracture of the metallic components after UKA is a rare complication. The Swedish Knee Registry reported that fracture of components after UKA occurred in 24 out of 1135 cases (2%), but detailed information, such as the parts involved and specific causes, were not described . ManzottMost previous reports of fracture of the metallic components in the literature occurred in the femoral component . There iIn the present case, the fracture site was the boundary between the distal and oblique posterior surfaces of the femoral component, which is the thinnest part of the femoral component. In addition, the metallic fracture site was, to some extent, distant from a peg. This is in contrast to a finding by Manzotti et al.  in whichFracture of the metallic femoral component after UKA is a rare complication associated with modern UKA prostheses. The present report indicates that implant loosening due to insufficient crimping of the femoral component using cement and excessive loading can cause fracture of a femoral component even with a modern implant with a better design. Thus, a careful procedure is necessary at the time of implantation with cement.


Dipeptide Gly‐Sar and CaSR agonist spermine markedly raised [Ca2+]cyt in villous enterocytes, which was abolished by NPS‐2143, a selective CaSR antagonist and U73122, an phospholipase C (PLC) inhibitor. Apical application of Gly‐Sar induced a jejunal short‐circuit current (Isc), which was reduced by NPS‐2143. CaSR expression was identified in the lamina propria and on the basal enterocyte membrane of mouse jejunal mucosa in both WT and −/−Slc15a1 animals, but Gly‐Sar‐induced [Ca2+]cyt signaling was significantly decreased in −/−Slc15a1 villi. Clotrimazole and TRM‐34, two selective blockers of the intermediate conductance Ca2+‐activated K+ channel (IKCa), but not iberiotoxin, a selective blocker of the large‐conductance K+ channel (BKCa) and apamin, a selective blocker of the small‐conductance K+ channel (SKCa), significantly inhibited Gly‐Sar‐induced Isc in native tissues. We reveal a novel CaSR‐PLC‐Ca2+‐IKCa pathway in the regulation of small intestinal dipeptide absorption, which may be exploited as a target for future drug development in human nutritional disorders.Although absorption of di‐ and tripeptides into intestinal epithelial cells occurs via the peptide transporter 1 ), the detailed regulatory mechanisms are not fully understood. We examined: (a) whether dipeptide absorption in villous enterocytes is associated with a rise in cytosolic Ca +/dipeptide absorption rates in the small intestine depend on the maintenance of a negative membrane potential rather than the transmembrane proton gradient. What this study adds: The CaSR mediates intestinal dipeptide absorption through the Ca2+/IKCa/hyperpolarization mechanism Clinical significance: CaSR‐PLC‐Ca2+‐IKCa pathway could be exploited as a potential target in human nutritional disorders.What is already known: The H Althoug+/dipeptide absorption at low and high luminal pH, and that sustained H+/dipeptide absorption rates depend on the maintenance of a negative membrane potential rather than the transmembrane proton gradient. When the membrane depolarization by the inhibition of Na+/K+‐ATPase was initiated in mouse jejunum, a major reduction in Gly‐Sar‐induced Isc response was observed  translocation  was employed to assess changes in cytosolic free Ca2+ concentrations ([Ca2+]cyt) in native microdissected microvilli of the CAG‐TN‐XXL and Slc15a1−/−‐CAG‐TN‐XXL transgenic mouse, which encodes a genetically anchored calcium‐sensing protein TN‐XXL , peptides  or Merck , if not indicated otherwise. Gly‐Sar, spermine, U73122 were purchased from Sigma . Apamin was purchased from Sigma . TRAM‐34 was purchased from MCE . NPS‐2143 was purchased from Tocris . Iberiotoxin and Clotrimazole were purchased from Tocris . Mouse anti‐CaSR antibody was purchased from ThermoFisher . Polyclonal rabbit anti‐Actin antibody was purchased from Abcam . The human colon cancer cell lines SW480 and SW620 were purchased from the Chinese Academy of Sciences in 2012. All cell lines were kept frozen in liquid nitrogen and after they were thawed, less than 20 of passages were used for 3 months in the present experiments.2.2Slc15a1−/− and Slc15a1+/+ CAG‐TN‐XXL mice. The mice were bred in the animal research institute of Hannover Medical School on the C57/B6 background and were genotyped as recommended   was removed and immediate placed in ice‐cold Ringer's solution . The jejunum was sliced into 0.5 cm sections and each section was opened along the mesenteric border. One piece of tissue was transferred on the cooled stage of a dissecting microscope, and individual villi were detached from the intestine by snapping them off from the mucosa with sharpened microdissection tweezers. Care was taken not to damage the apical part of the villi. The villi were attached to a glass coverslip coated with Cell‐Tak adhesive. The villi were fixed on the coverslip with a polycarbonate membrane  in a custom‐made perfusion chamber, and perfused with prewarmed (37°C) O2‐gassed buffer A . Buffer B, in which 20 mM Gly‐Sar, replaced 10 mM NaCl. The FRET‐based biosensor TN‐XXL composed of two fluorescent proteins, the cyan fluorescent protein (CFP) and the Citrine cp174 acting as donor (D) and acceptor (A), respectively. Ca2+‐imaging was done at a Zeiss LSM 780 with the acquisition settings as follows: 40x/1.2 NA water immersion objective, excitation 440 nm, online fingerprinting mode, reference spectra obtained from single fluorophores, 300 time steps, 10s interval. The acceptor/donor ratio (A/D ratio) was used as a readout of the TN‐XXL as measure for [Ca2+]cyt.In order to assess cyt  in villous enterocytes isolated from the jejunum of CAG‐TN‐XXL mouse , a commonly used CaSR activator, also caused a strong increase in [Ca2+]cyt, which was significantly inhibited by the CaSR inhibitor NPS‐2143 (30 μM), and U73122 (20 μM), a widely employed PLC inhibitor , and U73122 (20 μM)   Figure b. Howevee Figure . As in t3.32+]cyt plays a critical role in modulating intestinal epithelial ion transports . However, Ca2+ removal (Ca2+ omission plus 100 μM EGTA) from serosal side or both sides abolished Gly‐Sar‐stimulated jejunal Isc . In contrast, Ca2+ removal from mucosal side failed to alter Gly‐Sar‐induced Isc  of NPS‐2143, a selective CaSR antagonist , to both sides of the tissues. As shown in Figure 2+‐activated K+ channel (IKCa), significantly inhibited the Gly‐Sar‐induced Isc. However, neither Iberiotoxin (100 nM) a selective blocker of large‐conductance K+ channel (BKCa), nor Apa min (100 nM) a selective blocker of small‐conductance K+ channel (SKCa) evoked any obvious effect. These data suggest that IKCa rather than BKCa and SKCa are involved in H+/dipeptide absorption.The jejunal mucosal tissues were equilibrated in Ussing chambers for 30 min, basal 3.6Ca channels, BKCa (KCNMA1), SKCa (KCNN3), and IKCa (KCNN4), have been identified to be expressed in gastrointestinal epithelia and involved in intestinal absorptive and ion transport  that occur during H+/dipeptide uptake, and the strong autofluorescence of enterocytes in the blue spectral range. We therefore made use of the CAG‐TN‐XXL mouse, which expresses a genetically encoded FRET‐based biosensor for Ca2+, namely a genetically optimized troponin C, TN‐XXL to display an increased signal strength in the low‐calcium regime , peptides  family, plays a critical role in maintaining CaIsc response to Gly‐Sar was significantly reduced by the exclusively serosal addition of either Ca2+ chelator EGTA or CaSR antagonist. This suggests that the stimulation of the CaSR by addition of Gly‐Sar to the luminal bath occured beyond the enterocyte brush border membrane, in which PEPT1 resides. Intestinal CaSR expression has been localized to the apical and basolateral membrane of enterocytes (Cheng, Okuda, Hall, Geibel, & Hebert, In this study, we also observed that the jejunal −/−Slc15a1‐CAG‐TN‐XXL mouse, the percentage of reactive villous enterocytes were significantly decreased compare to CAG‐TN‐XXL. Previous reports suggested that 70% uptake of Gly‐Sar was via transecellular pathway in WT mice, the remaining 30% could be absorbed via paracellular pathways, passive diffusion or endocytosis (Chen et al., −/−Slc15a1‐CAG‐TN‐XXL mouse, we still observed calcium response, suggesting that CaSR is also involved in PEPT1‐independent increase of cytosolic calcium by Gly‐Sar. Wenzel et al. reported that PEPT1‐mediated cefixime uptake into human intestinal epithelial cells was increased by nifedipine, a putative CaSR agonist (Wenzel, Kuntz, Diestel, & Daniel, Gly‐Sar is a nonhydrolyzable dipeptide and therefore exclusively absorbed into enterocytes by PEPT1. Of the di‐ and tripeptides absorbed during a meal by PEPT1, the majority are further hydrolyzed and exported as aminoacids, but a fraction is also exported as dipeptides by as yet molecularly undefined carriers (Terada & Inui, +‐coupled electrogenic dipeptide uptake in the native small intestine mediated by PEPT1 was solely dependent on membrane potential, irrespective of extracellular pH. It is well known that the opening of the K+ channels lead to hyperpolarization of the cells, providing a driving force for HCO3‐ secretion, dipeptide absorption and Cl‐/HCO3‐ exchangers, moreover keeping the potassium channel open requires only a brief calcium transient signal (Chen et al., Ca channels significantly inhibited Gly‐Sar‐induced Isc, indicating that the IKCa channels are involved in the Gly‐Sar absorption. IKCa channels are functionally expressed on the basolateral side of duodenal epithelial cells and regulate intestinal transepithelial Cl‐ and HCO3‐ secretion (Dong et al., 2+]cyt may induce hyperpolarization via stimulation of IKCa, a process that increases the driving force for H+‐coupled electrogenic dipeptide absorption. This concept is depicted in Figure 2+‐dependent activation of PKC isoforms with subsequent effects of PEPT1 trafficking (Brandsch, Miyamoto, Ganapathy, & Leibach, It is unclear which mechanism is responsible for the importance of CaSR‐mediated signaling for Gly‐Sar uptake. Earlier work has demonstrated that the H2+]cyt in intestinal epithelial cells via CaSR activation. It is followed by activation of the basolateral IKCa channels by Ca2+ signaling and results in membrane hyperpolarization to promote further dipeptide absorption through a positive feedback loop. CaSR activation also triggers intracellular calcium release from the endoplasmic reticulum. Thus, CaSR‐induced Ca2+ signaling might sustain K+ channel gating to promote peptide absorption. In adults, the calcium absorption rate is 1,000 mg per day (Bronner & Pansu, 2+ can bind the calcium‐buffering proteins, while some contributes refilling of ER calcium store and promotes the CaSR‐PLC‐Ca2+‐IKCa signaling cascade. Therefore, CaSR‐PLC‐Ca2+‐IKCa signaling pathway may be involved in intestinal nutrient absorption, suggesting the imbalance of this pathway may contribute to human nutritional disorders.What is the physiological relevance of this study? Food nutrients, such as dietary dipeptides and L‐amino acids, can raise [CaThe authors have no conflict of interest to disclose.J.X., A,Z., B.R., S.Y., O.G., H.D., B.G., E.P., U.S., and H.D. conceived and designed research; J.X., A,Z., B.R., performed experiments; J.X., B.R., and A,Z., interpreted results of experiments. J.X., A,Z., and H.D., analyzed data. J.X. and A,Z prepared figures. J.X. and H.D. drafted the manuscript. H.D. and U.S. edited and revised the manuscript. J.X., E.P., H.D., and U.S. approved the final version of the manuscript. Click here for additional data file.


Here we determined the X-ray crystal structure of a Ca2+-dependent AFP (jsAFP) from Japanese smelt, Hypomesus nipponensis, in both Ca2+-bound and -free states. Their overall structures were closely similar (Root mean square deviation (RMSD) of Cα = 0.31 Å), while they exhibited a significant difference around their Ca2+-binding site. Firstly, the side-chains of four of the five Ca2+-binding residues  were oriented to be suitable for ice binding only in the Ca2+-bound state. Second, a Ca2+-binding loop consisting of a segment D94–E99 becomes less flexible by the Ca2+-binding. Third, the Ca2+-binding induces a generation of ice-like clathrate waters around the Ca2+-binding site, which show a perfect position-match to the waters constructing the first prism plane of a single ice crystal. These results suggest that generation of ice-like clathrate waters induced by Ca2+-binding enables the ice-binding of this protein.Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca Tf), without a significant increase of the melting point (Tm) . N. N2+-dep2, and held for 2 h to induce ice-binding of jsAFP. Following this immersion, three red-colored ellipses were observed on the ice hemisphere  ,272, and2+-bound state was determined with preparation of its crystal in a solution composed of 0.1 M sodium acetate buffer (pH 7.0), 0.2–0.25 M ammonium sulfate, 8% PEG 3350, and 2 mM CaCl2. For determination of the structure in the Ca2+-free state, the crystal was soaked into the above solution without CaCl2 for which pH was adjusted to 3.0, since 1 mol of Ca2+ is detached from jsAFP in the acidic condition. The jsAFP crystals were affected by neither pH nor Ca2+. The diffraction data was collected at 1.06 and 1.25 Å resolution for Ca2+-bound and -free states, respectively.The X-ray structure of jsAFP in the Ca2+-bound state, a structural model obtained with the molecular replacement method showed its clear density map including 1 mol of Ca2+. This model was further refined with including this Ca2+ ion, thereby elucidating the final structural model whose B-factor (Å2) is comparable between the backbone and side-chain atoms. This final model consists of 126 amino acid sequence from E3 to T128 as well as 289 water molecules with R factor of 0.154 and Free R factor [2+-bound jsAFP is composed of two alpha-helices (α1 and α2), eight beta-strands (β1–β8) and four loop regions  (2+-dependent hAFP (RMSD of Cα = 0.46Å) and of the Ca2+-independent BrAFP (RMSD of Cα = 0.71 Å) [2+ ion was located at the molecular surface between β7 and a loop segment L3–L4 of jsAFP, and was coordinated with the side-chain oxygen atoms of Q92, D94 E99, D113, D114, a backbone oxygen atom of D114, and an oxygen atom of a water molecule.For jsAFP in the CaR factor  of 0.174D94–E99) A. The mo 0.71 Å) ,27. A Ca2+ in the jsAFP structure is highly similar to that in the hAFP structure. Among the six proline residues of jsAFP, P93 located between the two Ca2+-binding loops L3 and L4 adopts cis-conformation. This is conserved in all known three-dimensional structures of C-type lectins [2+-binding site. The crystal structure of Ca2+-free jsAFP was determined to be 1.25 Å resolution (PDB ID: 6JK5), which consists of 125 amino acid sequence from C4 to T128 as well as 237 water molecules with an R factor of 0.18 and a free R factor [2+-bound state, which allowed an in-depth comparison between the two crystal structures. The similarity of their overall construction was indicated by a significantly low RMSD value (0.31 Å) between the two forms calculated using their 125 Cα-atom positions. Structural changes were only observed near the Ca2+-binding site  along the amino acid sequence between the Ca2+-bound and -free structures of jsAFP. A rapid molecular motion tends to raise the B factor of that segment, a significant increase evaluated for the Ca2+-binding loop L4 (D94–E99) suggests that this loop possesses a flexible nature only in the Ca2+-free form.The coordination geometry of Ca lectins , so thatR factor  of 0.247ing site B. An eleund form B left. Tposition B right. t 353 nm . Figure 2+-binding loop (L3 and L4) of jsAFP with that of the two members of C-type lectin, tetranectin3 (TN3) and a mannose-binding protein (MBP) [2+-free and -bound states (2+ is largely moved away from interior (cyan) to outside (orange) when Ca2+ ions are released. The Ca2+-binding loop of MBP associated with 2 mol of Ca2+ also becomes opened by the removal of Ca2+ (cyan→orange). In addition, cis-proline residue located at the junction between L3 and L4 of MBP, which corresponds to P93 of jsAFP, is isomerized to a trans-conformation by the Ca2+-removal. The loop region is probably pinned by the calcium ions in both TN3 and MBP, so that the region becomes free and moved toward outside when the ions are released. Significantly, the backbone conformation of the Ca2+-binding loop of jsAFP including cis-conformation of P93 is not virtually affected by removal of Ca2+. The reason for such structural holding might be due to construction of two disulfide-bonds, Cys69–Cys100 and Cys89–Cys111, which are highly conserved in type II AFPs but not in the other C-type lectins  ,44. For d states A. In con lectins B. For ex2+-binding loops) are exposed to the solvent in both Ca2+-bound and -free states. A total of 289 and 237 hydration waters were observed on the surface of the Ca2+-bound and -free structures of jsAFP, which are represented by spheres colored with cyan and orange, respectively , is located in an area indicated with a hatched ellipse, on which it detected many hydration waters (cyan) only for the Ca2+-bound state. Significantly, a total of 16 waters are organized into an ice-like arrangement on the putative IBS (I~PV) and one hexagonal ring (HI) that are next to each other, in which a few oxygen atoms of the side-chain groups of jsAFP are also involved. Among them, the waters labeled 1, 4, 5, 6, 10, 12, and 14 directly anchor to the protein, while the others form second or third hydration layer on the protein. In contrast, no robust formation of such organized waters was detected in the Ca2+-free form  is lower than that of the Ca2+-free form (orange line), suggesting that 1 mol of Ca2+-binding rigidifies the overall structure of jsAFP. The hAFP in the Ca2+-free state was easily digested by trypsin while being less digested in the Ca2+-bound state [2+-free state. For a Ca2+-binding loop (L4) from D94 to E99A of jsAFP, a significant difference of the B-factor was detected (2+. This loop region becomes more flattened, and a relevance between the flatness and IBS construction was also suggested [2+-bound jsAFP, suggesting that the flexible Ca2+-binding loop might be unfavorable for construction of the hydrogen bonding networks on this region. Indeed, the side-chains of D94, T96, T98, and E99 in the Ca2+-free jsAFP are not hydrogen-bonded to any water molecule.It was reported that structural rigidity and hydrophobicity of IBS are key determinants to forming the semi-clathrate waters . The typnd state , which adetected C, implyiuggested . The ice2+ ion in the jsAFP structure plays a role to neutralize the electric charge of the acidic residues of the IBS, which increases its hydrophobicity to lead and organize the polypentagonal ice-like waters. It is interesting that Ca2+-binding to jsAFP not only causes a slight conformational change of the side-chains near the IBS, but also fine-tunes the protein’s electricity and hydrophobicity to modify the hydration manner, leading to generating the ice-like waters to bind the host protein to an ice crystal surface.Hydrophobicity of the molecular surface is also relevant to the protein hydration. Talon et al., 2014 reported that hydrophobic protein surface tends to be surrounded by the polygonal semi-clathrate waters, which avoids its exposure to the solvent . They sh2+-dependent type II AFP from Japanese smelt denoted jsAFP, and made a structural comparison between its Ca2+-bound and -free forms to elucidate its ice-binding mechanism via Ca2+ ion. The obtained data revealed that binding of Ca2+ does not affect overall structural motif of jsAFP but alters its side-chain conformation as well as its rigidity of the Ca2+-binding loop, facilitating a formation of the polypentagonal ice-like waters on the IBS. These semi-clathrate waters exhibited perfect position-matches to the waters constructing the 1st prism plane, which was assigned to a target ice plane of jsAFP through the observation of the FIPA pattern on a single ice crystal hemisphere. This is the first report of Ca2+-induced ice-like water formations on the surface of AFP, which may suggest that AFPs inevitably use the ACW mechanism for its ice binding function.In the present study, we determined the crystal structure of a Ca


Dehalococcoides spp. However, most genes have not been functionally characterized. Biochemical studies on RDases have been hampered by difficulties encountered in their expression and purification. In this study, we have expressed, purified and characterized RdhA of RDase for tetrachloroethene (PceA) from Geobacter sp. PceA was expressed as a fusion protein with a trigger factor tag in Escherichia coli. PceA was purified and denatured in aerobic condition. Subsequently, this protein was refolded in the presence of FeCl3, Na2S and cobalamin in anaerobic condition. The reconstituted PceA exhibited dechlorination ability for tetrachloroethene. UV-Vis spectroscopy has shown that it contains cobalamin and Fe-S clusters. Since this method requires anaerobic manipulation only in the reconstituting process and has a relatively high yield, it will enable further biochemical studies of RDases.Reductive dehalogenase (RDase) consists of two parts, RdhA and RdhB. RdhA is the catalytic subunit, harboring a cobalamin cofactor and two Fe–S clusters. RdhA is anchored to the cytoplasmic membrane via the membrane anchoring subunit, RdhB. There are many genes encoding RDases in the genome of organohalide-respiring bacteria, including  Dehalococcoides, Dehalogenimonas, Dehalobacter, Desulfitobacterium, and Sulfurospirillum, as well as the organohalide-respiring members of the Deltaproteobacteria. Organohalide-respiring bacteria play critical roles in anaerobic bioremediation of sites contaminated by organohalides  in Bacillus megaterium . After purification and denaturation in 8 M urea in aerobic conditions, PceA was reconstituted with Fe and cobalamin in anaerobic condition. Thus, the reconstituted TF-PceA-Strep exhibited RDase activity on PCE.In the present study, we tried functional expression and characterization of PceA from pceA gene was obtained by PCR from the metagenome DNA of PCE dechlorinating bacterial consortium using the primers (5′-ATG GAT CGT AGA GAT TTT TTT AAA AAG GCA GC-3′ and 5′-CTA TGC CTT GTT CCA GAA GTC CG-3′). The amplified DNA was cloned into the T-vector pMD20  and subjected to DNA sequencing.The pceA was amplified using the primers (5′-CCC AAG CTT ATG GAT CGT AGA GAT TTT TTT AAA AAG G-3′ and 5′-CCC TCG AGC TAT GCC TTG TTC C-3′) and cloned into the HindIII/XhoI site of pET23b  (pET23b_PceA).The full-length To express PceA as a fusion protein with TF, the gene was amplified with the primers (5′-CGA GCT CAT GGA TCG TAG AGA TTT TTT TAA AAA GG-3′ and 5′-CCC TCG AGC TAT GCC TTG TTC C-3′), and subcloned into the SacI/XhoI site of pCold TF vector  (pCold TF-PceA). Then, the Strep-tag sequence was inserted at the C-terminus by the QuikChange method using the primers (5′-CGG ACT TCT GGA ACA AGG CAT GGA GCC ATC CGC AGT TTG AAA AGT AAG TCG ACC TGC AGT CTA GAT AG-3′ and 5′-CTA TCT AGA CTG CAG GTC GAC TTA CTT TTC AAA CTG CGG ATG GCT CCA TGC CTT GTT CCA GAA GTC CG-3′) (pCold TF-PceA-Strep).E. coli BL21 star (DE3). The transformed cells were grown in Luria-Bertani medium containing 100 μg/ml ampicillin at the specified temperatures. The cells were harvested by centrifugation at 5000 rpm for 15 min at 4°C.Thus, the prepared plasmids  were used for the transformation of Approximately 3 g wet weight of TF-PceA-Strep was re-suspended in 30 ml of suspension buffer . After cell disruption using an ultrasonic disruptor , the suspension was centrifuged for 15 min at 14,000 rpm. The supernatant was discarded, and the pellet was resuspended in 15 ml of the denaturation buffer (suspension buffer + 8 M urea). Following another round of sonication, the suspension was stirred on ice for 1 h to solubilize the pellet. Centrifugation for 15 min at 4°C at 14,000 rpm was performed to remove remaining insoluble material.The supernatant containing denatured TF-PceA-Strep was loaded onto the Ni-affinity column , which had been washed with five column volumes of the denaturation buffer and eluted with four column volumes of 250 mM imidazole in the denaturation buffer.3) and S buffer  were added up to 50 mol excess of Fe and S to TF-PceA-Strep. After 90 min incubation with stirring, cyanocobalamin was added to a final concentration of 10 mg/ml. Next, TF-PceA-Strep solution was applied to a PD-10 column  equilibrated with the refolding buffer . The fractions containing reconstituted TF-PceA-Strep were collected.Next, the reconstitution of TF-PceA-Strep was performed under anaerobic conditions. All buffers were purged with 99.99% Ar and placed in the anoxic glove box before use. Denatured TF-PceA-Strep was mixed with the reduction buffer  to 5 mM of DTT, and incubated for 30 min with stirring. Then, Fe buffer  citrate as the electron donor and 0.4 mM methyl viologen as the electron mediator was added. The reaction was started by injecting PCE to approximately 100 μM PCE. Headspace samples (100 μl) were injected into the gas chromatograph  equipped with a DB-624 column  and a flame ionization detector.The band for TF-PceA-Strep in SDS-PAGE was excised and applied for the treatment by In-Gel Tryptic Digestion Kit . LC–MS/MS analysis of the digested peptides was performed using reversed-phase LC interfaced with a Q-TOF mass spectrometer . The digest peptides were separated using a PEGASIL ODS SP300-3 column  eluted with a linear gradient of 0–100% buffer B (100% acetonitrile and 0.1% trifluoroacetic acid) in buffer A (0.1% trifluoroacetic acid in water) at a flow rate of 0.04 ml/min. MS and MS/MS data were acquired using the data-dependent top five method. The resulting MS/MS data were searched against the sequences of TF-PceA-Strep, using BioTools .UV-Vis absorbance spectra were recorded with a Varian CARY 50 UV-VIS Spectrophotometer (Agilent Technologies) in aerobic condition.Co and Fe content was analyzed by graphite furnace atomic absorption spectrometry (GFAAS). We used the Polarized Zeeman Atomic Absorption Spectrophotometer ZA3000  for GFAAS. At first, we made the calibration curves using the standard solutions of Fe and Co at the concentration of 0, 2.5, 5.0, 10, or 15 ppb in the Refolding Buffer . Next, the reconstituted TF-PceA-Strep was diluted with 2% nitric acid. The diluted samples were applied for GFAAS analysis. The average values of triplicate measurements were used.The secondary structure of the refolded TF-PceA was analyzed by CD. Protein samples were prepared in 2 mM Tris-HCl, pH 8.0 at 8.9 μM and then filtrated with cellulose acetate filter (0.2 μm). CD spectrum with a wavelength range of 205–260 nm was measured at 20°C by CD spectrometer  using a 2 mm optical path length cuvette.cis-1,2-dichloroethene (cis-DCE) . The 16S rRNA gene analysis of the metagenome showed the presence of Dehalobacter sp., Geobacter sp., Sulfurospirillum sp., Clostridium sp. and Bacteroides sp. Among these species, we speculated that Dehalobacter sp. or Geobacter sp. is responsible for dechlorination of PCE. Next, we attempted to amplify pceA genes from the metagenome using various primers designed from pceA genes of Dehalobacter sp. and Geobacter sp. We could obtain the full-length pceA gene, which exhibits significantly high sequence identity with that of G. lovleyi SZ . The Nucleotide sequence is available in the DDBJ/EMBL/GenBank databases under the accession number, LC342077. G. lovleyi SZ was obtained from the non-contaminated creek sediment microcosms based on their ability to derive energy from acetate oxidation coupled to PCE-to-cis-DCE dechlorination  sites are also conserved. The first ISB, CKNCKKCADACP, corresponds to the conserved consensus sequence CXXCXXCXXXCP, found in bacterial ferredoxins  to rination . The putredoxins . The secredoxins . HoweverGeobacter PceA in E. coli, the Geobacter pceA was cloned into the pET23b vector (pET23b-pceA) and used to transform E. coli BL21 star (DE3). PceA was obtained as an aggregate in the insoluble fraction . Next, we tried to express PceA as a fusion protein with the trigger factor tag (TF-PceA). TF-PceA was obtained as a soluble protein at the induction of protein expression at 18°C . E. coli cells expressing TF-PceA were collected and disrupted by the lysozyme treatment at the anaerobic conditions in a glove box. The total cell lysate was applied for dechlorination assay for PCE. Unexpectedly, no PCE dechlorination activity was observed. We further tried to purify TF-PceA in aerobic conditions using the histidine tag attached to TF tag. However, TF-PceA was subjected to protease digestion during purification by Ni affinity chromatography. Next, we added Strep-tag at the C-terminus (TF-PceA-Strep). TF-PceA-Strep was purified by affinity chromatography using Strep-tactin column and size exclusion chromatography. In this case, no protease digestion was observed. In the mass spectroscopic analysis, the proteolytic peptide fragments covered approximately 40% of TF-PceA-Strep . Since UV-Vis spectrum of the purified TF-PceA-Strep lacked the peak for cobalamin, we concluded that the active center was not correctly formed in TF-PceA-Strep.To express the E. coli, it is difficult to purify, because all procedures must be performed in anaerobic conditions. Thus, we decided to reconstitute active PceA in vitro. Since it was difficult to reconstitute PceA in vitro, we used TF-PceA-Strep. We expressed TF-PceA-Strep as an insoluble protein by culturing recombinant E. coli at 37°C. Then, the precipitated proteins were solubilized by buffer containing 8 M Urea. TF-PceA-Strep was purified by nickel affinity chromatography in the presence of 8 M urea. The denatured TF-PceA-Strep was applied for reconstitution in the anaerobic glove box. Denatured TF-PceA-Strep was mixed with DTT, FeCl3, Na2S and cobalamin in denaturation buffer, and the mixture was loaded onto a PD-10 desalting column equilibrated with the refolding buffer. TF-PceA-Strep was expected to reconstitute by removing urea, and unincorporated excess cofactors were also removed. CD spectrum shows that the reconstituted TF-PceA-Strep takes the folded conformation .We speculated that although active PceA was obtained in Figure 3, in the presence of TF-PceA-Strep, PCE decreased with the increase of TCE and cis-DCE. However, a decrease in PCE and the appearance of TCE and cis-DCE were not observed in the control experiments using a protein unrelated to reductive dehalogenation, Hsp104 from a thermophilic fungus.Thus, prepared reconstituted TF-PceA-Strep was used for the dechlorination assay for PCE. The reconstituted TF-PceA-Strep was mixed with PCE in the presence of Ti(III) citrate as the electron donor and methyl viologen as the electron mediator. As shown in Figure 4). To obtain the quantitative data, the metal content of the reconstituted TF-PceA-Strep was analyzed by GFAAS (Table 1). The molar ratio of TF-PceA-Strep:Co:Fe was 1:1.4:10. This ratio was close to the theoretical value of 1:1:8, suggesting that TF-PceA-Strep and cofactor coexist with values close to the theoretical values.Finally, we examined the reconstitution of the catalytic centers by UV-Vis spectrophotometry and metal analysis. Because of the apparatus limitation, we performed UV-Vis spectrophotometry in aerobic conditions. Before reconstitution, no peaks derived from the cofactor were detected. Alternatively, peaks attributed to each cofactor were detected from the sample after reconstruction; [4Fe–4S] at 420 nm, Co(I) at 360 and 550 nm, and Co(II) at 310 nm  tag, and purified under anaerobic conditions  is approximately 37%. The two ISB motifs are conserved with that of PceA from Dehalobacter sp. E1. However, cobalamin binding motif is not well-conserved. Because Geobacter sp. and Dehalobacter sp. seemed to be responsible for dechlorination of PCE in the original bacterial consortium, we concluded that the gene was originated from a Geobacter sp.In this study, we tried to express and characterize the PceA. The cis-DCE via TCE. UV-Vis and GFAAS suggested binding of TF-PceA-Strep and a cofactor. The measured metal content of the reconstituted sample was partially larger than the theoretical value. This result is probably observed because the separation of free Fe and cobalamin by PD-10 is not sufficient.We expressed PceA as a fusion protein with the trigger factor tag and also the Strep-tag (TF-PceA-Strep). TF-PceA-Strep was denatured by buffer containing 8 M urea and purified by affinity chromatography using the histidine tag in trigger factor tag. Although TF-PceA-Strep was only partially purified in this study, it is possible to perform further purification using different types of chromatography. The reconstitution of TF-PceA-Strep was performed by size exclusion chromatography using a buffer containing cofactors in anaerobic conditions. Since TF-PceA-Strep can refold easily, this process requires only a small column. The reconstituted TF-PceA-Strep could convert PCE to We have established a heterologous platform to produce the TF-PceA-Strep enzyme, which can be reconstituted in its active form. It was expected that the function and structure of RDase would be clarified by the crystallization of RDase under anaerobic conditions. Further study is needed to understand all of the components related to the synthesis and activity of RdhA, with continued future research efforts investigating the accessory genes and their products, such as transcriptional regulators , anchoring proteins (RdhB or PceB), and maturation proteins . This platform may be employed for obtaining recombinant proteins with the same characteristics as RDase.RNA performed all data collections. TOB, YH, TOG, and KN assisted the data collection. RNO constructed PCE dechlorinating bacterial consortium and assisted data collection. MY designed the study and wrote the manuscript.The 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.


Overactive bladder (OAB) and mixed urinary incontinence (MUI) are significant problems worldwide. Their broad definition makes them difficult to diagnose; therefore, specialists need a tool to confirm diagnosis. The Overactive Bladder Symptom Score (OABSS) is used in the objective diagnosis of OAB. We aimed to develop and evaluate the effectiveness of OABSS for patients in Poland suffering from OAB and MUI and to correlate it with UDI-6 and IIQ-7.n = 290); OAB (n = 285) and MUI (n = 249) were confirmed by medical history and urodynamic study. Of the subjects, 821 women completed the Polish version of OABSS on two separate visits: weeks 0 and 2. In addition, they undertook UDI-6 and IIQ-7 during Week 2. The Cronbach’s alpha (α) was used to estimate the internal consistency. Scores were compared using the intraclass correlation coefficient (ICC).A total of 824 women suffering from urinary incontinence (UI) aged between 18 and 75 years were included. SUI (p < 0.0005) between mean scores of OABSS among patients from the study groups OAB-SUI and MUI-SUI. We did not observe statistically significant differences between patients from the MUI and OAB groups (p > 0.11). Analysis also did not show statistically significant differences between visits.We observed statistically significant differences ; = 0.9 (OAB); = 0.82 (MUI). In all groups, test–retest reliability was excellent; ICC was >0.99.The Polish version of the OABSS is a reliable tool for females suffering from UI. However, OABSS does not distinguish patients with MUI from patients with OAB. Overactive bladder (OAB) and mixed urinary incontinence (MUI) are significant problems worldwide. Indeed, the prevalence of OAB was expected to increase about 20.1% from 2008 to 2018 . Hence, Currently, the diagnostic process of OAB consists of: medical history (MH), physical and neurological examination, questionnaires and/or bladder diary (BD), urine analysis and urodynamic study (UDS) . UDS is The Overactive Bladder Symptom Score (OABSS) has been developed to express all of the OAB symptoms in a single result. Four symptoms addressing day- and night-time frequency, urgency, and urgency incontinence are scored in the Homma OABSS . The BlaIn our work, the survey was designed to validate the efficacy of the seven-question OABSS for the Polish population by establishing the validity, test–retest reliability and internal consistency of a professionally translated questionnaire. This was done to secure a valid instrument for urinary incontinence (UI) diagnosis and to correlate it with UDS, the Urogenital Distress Inventory (UDI-6), and the Incontinence Impact Questionnaire (IIQ-7).Three independent translators translated the original OABSS into Polish. A native speaker back-translated it into English to ensure conceptual equivalence with the original OABSS version. Ten women suffering from UI (confirmed by urodynamics) were tested using the Polish version of the questionnaire. No major issues were observed, and minor problems that arose were straightened out.A total of 824 women with UI aged between 18 and 75 years, who met the inclusion criteria, were chosen from patients attending the Outpatient Clinic between June 2017 and December 2018. After signing the informed consent, socio-demographic data such as age, parity, BMI, and menopausal status were taken. Polish was the native language of all patients within the study population. Of the study group, recruitment medical examination/case history and urodynamic investigation performed during visit week 0 showed that 290 patients had SUI, 285 had OAB, and 249 had MUI. All patients completed the OABSS questionnaire at baseline (visit week 0). Moreover, 821 women repeated the OABSS and 824 completed the UDI-6 and IIQ-7 questionnaires after 14 days. Twenty-three patients who did not reappear in week 2 were excluded from evaluation. None of the patients received treatment between visits. In this part of the study, only the answers of patients who fully completed the questionnaires were taken into account. The local bioethics committee approved the study.The sample size was established based on Comfrey and Lee’s suggestion that 50 is very poor, 100 is poor, 200 is fair, 300 is good, 500 is very good, and 1,000 or more makes for an excellent sample size [Face/content validity was assessed by obtaining feedback from a group of 10 women suffering from UI, and from 3 doctors taking part in the study who were specialists in the urogynecology field. They reviewed the seven-question OABSS questionnaire and clarified the translation.To evaluate the factor structure and construct the validity of the OABSS, principal component analysis (PCA) using VARIMAX rotation was conducted for all seven questionnaire items. The data from the entire sample group at day 0 were analyzed. PCA results were also confirmed by Spearman’s correlations among OABSS items.The final Polish version of the OABSS was subsequently administered to Polish-speaking women assessed to have UI at university-based urogynecology clinics in Poland. Polish versions of the UDI-6 and IIQ-7 were also administered to evaluate the criterion validity of the Polish OABSS. The results of UDI-6 and IIQ-7 application were compared using Pearson’s correlation coefficient to assess the criterion validity.The internal consistency of the OABSS questionnaire was estimated by way of Cronbach’s alpha coefficient (α). Here, a value greater than 0.7 indicates high reliability.Reliability was tested by applying the intraclass correlation coefficient (ICC), an index or repeatability, by utilizing statistical software R. Here, ICC at ≥0.7 was considered acceptable .p values less than 0.05 were considered significant. Significance of differences of means between studied groups was assessed using one-way ANOVA and the Tukey post hoc test.Statistical analysis was performed using STATISTICA version 13.1 software  in addition to open source R software . p valueDetailed characterization of patients included in the study is presented in Table The validation process shows that the Polish OABSS version has good face validity—no major troubles arose during the application and most respondents found it “comprehensive” and a “good” measure.The result of PCA using VARIMAX rotation indicated that the high degree of system variation  is explained by the first two components F1 and F2). F1 correlates strongly questions 1–6, whereas F2 correlates strongly only with question 7. The reason why not all of the questions constitute the first principal component (F1) was the fact that the last question, unlike the others, concerned the subjective feelings of the patient (Table . F1 corrp < 0.05). Here, item 7 was found to be poorly correlated with the other questions.In the study, a Pearson’s correlation coefficient between OABSS and UDI-6 of 0.676 and between OABSS and IIQ-7 of 0.545 demonstrates good convergent validity, with high correlation among OABSS items 1 to 6 (p < 0.0005) between mean scores among patients from the OAB-SUI and the MUI-SUI study groups and no statistically significant differences between patients from the MUI and OAB groups (p > 0.11). Moreover, analysis did not register important differences between visits.The total score of the OABSS ranges from 0 to 28. Table For UDI-6, a higher question response value indicates an increased level of disability; in IIQ-7, a higher score denotes diminished quality of life and enhanced severity of symptoms (maximum result of the questionnaire is 100) . ResultsTable The OABSS is an instrument used to assess patients with the OAB syndrome. It measures episodes of urination, urgency, and urgency incontinence. It is short (consisting of only seven questions) and simple. The original language version is considered reliable and is widely used. However, until this research, OABSS has never been applied in Poland or translated into Polish. Urgency is one of the symptoms of MUI, therefore, we used the new Polish version of OABSS in an attempt to distinguish between OAB and MUI.The Scientific Advisory Committee (SAC)—a nonprofit public service organization established to serve as a depository and distributor of high quality, standardized, health outcome measurement instruments—states that any instrument that measures health status must have the characteristics of a valid conceptual and measurement model. These include reliability, validity, responsiveness, interpretability, low respondent and administrative burden, being of comparable alternative forms, and holding appropriate cultural and language adaptation .internal consistency or reproducibility of research measures.Reliability is the p > 0.05); hence, OABSS cannot distinguish between OAB and MUI.Internal consistency refers to general agreement between different elements in the same test and is a means of estimating whether several items that purport to measure the same general structure, give similar results . To testIn addition to the OABSS, the patients of our study completed the UDI-6 and IIQ-7 questionnaires. The results showed statistically significant differences among patients from all study groups. It was previously stated that UDI-6 and IIQ-7 questionnaires are not the best option for confirming MUI diagnosis . Thus, wThe International Consultation on Incontinence Modular Questionnaire (ICIQ) Short Form is a diagnostic tool that evaluates the urinary tract symptoms and their influence on the quality of life (QoL) in women and men. Previous studies  show itsReproducibility (the possibility of the experiment or study being replicated) was tested by applying the ICC according to McGraw and Wong . The tesInternal validity or the accuracy of conclusions about whether one variable causes another was assessed using UDS (cystometry and uroflowmetry). We rightfully expected that SUI patients would score better in OABSS than did patients suffering from the other UI types Table .Based on the results of the study, the Polish version of OABSS is useful, but it cannot distinguish between OAB and MUI. The Polish OABSS is reliable, has very good psychometric validity, and can therefore be used by clinicians for preliminary screening for OAB.Urinary incontinence is a significant problem worldwide, and tools such as OABSS can help both specialists and nonspecialists in its diagnosis. OABSS, however, cannot distinguish between OAB and MUI.