In summary, this study shows that osteocytes expressed MENK, whereas the MENK expression was suppressed by compressive force via CTGF signaling. MENK downregulated nuclear translocation of NFATc1 probably by suppressing Ca2+ signaling in osteocytes and consequently inhibiting compressive force‐induced osteocyte apoptosis, followed by bone resorption. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.Mechanical stress stimulates bone remodeling, which occurs through bone formation and resorption, resulting in bone adaptation in response to the mechanical stress. Osteocytes perceive mechanical stress loaded to bones and promote bone remodeling through various cellular processes. Osteocyte apoptosis is considered a cellular process to induce bone resorption during mechanical stress‐induced bone remodeling, but the underlying molecular mechanisms are not fully understood. Recent studies have demonstrated that neuropeptides play crucial roles in bone metabolism. The neuropeptide, methionine enkephalin (MENK) regulates apoptosis positively and negatively depending on cell type, but the role of MENK in osteocyte apoptosis, followed by bone resorption, in response to mechanical stress is still unknown. Here, we examined the roles and mechanisms of MENK in osteocyte apoptosis induced by compressive force. We loaded compressive force to mouse parietal bones, resulting in a reduction of MENK expression in osteocytes. A neutralizing connective tissue growth factor (CTGF) antibody inhibited the compressive force‐induced reduction of MENK. An increase in osteocyte apoptosis in the compressive force‐loaded parietal bones was inhibited by MENK administration. Nuclear translocation of NFATc1 in osteocytes in the parietal bones was enhanced by compressive force. INCA‐6, which inhibits NFAT translocation into nuclei, suppressed the increase in osteocyte apoptosis in the compressive force‐loaded parietal bones. NFATc1‐overexpressing MLO‐Y4 cells showed increased expression of apoptosis‐related genes. MENK administration reduced the nuclear translocation of NFATc1 in osteocytes in the compressive force‐loaded parietal bones. Moreover, MENK suppressed Ca Osteocytes are the most abundant cells in bones and are embedded in bone matrices. Osteocytes have their dendritic processes through canaliculi to form an intercellular communication network via gap junctions. Osteocytes perceive mechanical stress as mechanosensors, transform into biochemical signals, and regulate bone remodeling by transmitting signals related to bone formation and resorption.Vertebrate skeletons are influenced by various kinds of mechanical stress during daily activities. Mechanical stress stimulates bone remodeling, which occurs through bone formation and resorption, resulting in bone adaptation to the mechanical stress.1 Ostenctions., 3 Osteorption., 5 Therefore, osteocytes are considered critical for osteoclastogenesis and bone resorption. Importantly, not only living osteocytes, but osteocytes undergoing apoptosis also promote bone resorption. We previously reported that compressive force induces osteocyte apoptosis in the alveolar bones during experimental tooth movement in mice and in cell culture of chick calvaria‐derived osteocytes. Our data suggest that osteocyte apoptosis occurs in response to compressive force and induces bone resorption. As to the mechanism of the compressive force‐induced osteocyte apoptosis, we revealed that connective tissue growth factor (CTGF) is a molecule that facilitates osteocyte apoptosis. However, the underlying molecular mechanisms of the compressive force‐induced osteocytes apoptosis are not fully understood.Osteocytes express receptor activator of nuclear factor kappa B ligand (RANKL), an essential factor for osteoclastogenesis.6 Therorption., 8 Impoorption., 10, 11eocytes., 13 Our The neuropeptide methionine enkephalin (MENK) is an endogenous opioid encoded by the proenkephalin (PENK) gene. It binds mainly to the delta opioid receptor (DOR) among three opioid receptors: mu, delta, and kappa. It is well known that MENK is expressed in nervous tissue, including the brain and spinal cord. MENK is also expressed in non‐nervous tissue, such as bone, heart, pancreas, and kidneys. Its analgesic property is a major function of MENK; moreover, MENK has other functions, including anti‐inflammatory effects and regulation of embryonic organ development. On the other hand, long‐term administration of opioids to alleviate cancer pain reduces bone mass, suggesting that opioids have a close relationship with bone remodeling. MENK is expressed in osteoblasts and mesenchymal stem cells, which are precursors of osteoblasts. In addition, MENK inhibits osteoblast differentiation. These findings suggest the inhibitory effect of MENK on bone formation regulating osteoblast differentiation during bone remodeling. However, the role of MENK in bone resorption is still unknown.Recent studies have demonstrated that neuropeptides play crucial roles in bone metabolism by regulating osteoblast and osteoclast function., 15, 16kidneys., 20, 21lopment., 23 On oblasts. In addi Conversely, activation of DOR suppresses apoptosis of neurons and hepatocytes. These studies suggest that MENK regulates apoptosis positively and negatively depending on cell type. This notion led us to hypothesize that MENK has a novel role in bone resorption by regulating osteocyte apoptosis in response to compressive force. Thus, we first analyzed the expression of MENK in osteocytes and its effect on apoptosis using the mouse model of compressive force loading to cranial bones that we have already established. Furthermore, we analyzed the molecular mechanisms of MENK in osteocyte apoptosis induced by compressive force. Nuclear factor of activated T cells (NFAT) induces apoptosis in various types of cells, such as pulmonary artery smooth muscle cells, neurons, and glomerular mesangial cells. Lu and colleagues reported that NFATc2 activation by MENK significantly induces apoptosis of rat C6 glioma cells. Therefore, we focused on NFAT to elucidate the MENK‐mediated mechanism in osteocyte apoptosis.McTavish and colleagues reported that PENK enhances apoptosis of human embryonic kidney (HEK) 293 cells and various human tumor cell lines.27 Contocytes., 29 Thel cells., 32, 33ad libitum access to fresh chow and water.Animal experiments were performed in accordance with the regulations for animal experiments and related activities at Tohoku University. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Tohoku University Environmental and Safety Committee. Mice were housed in groups of up to four animals in a cage and kept at 20°C to 26°C with 12‐hour light/dark cycles. They had B), fully covered by skin, and the incision was closed by suturing. In the control group, identical surgical procedures were performed without spring installation.Six‐week‐old male Institute of Cancer Research (ICR) mice  were anesthetized by i.p. injection of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg). A skin incision was made to expose the parietal bones. Two holes were made equidistant from a sagittal suture at the anteroposterior middle of the parietal bones using a round bur attached to a dental drill. The distance between the two holes was 5 mm. A spring that loads 0.2 N of compressive force onto the parietal bones was made by bending a 0.016‐inch diameter orthodontic beta–titanium wire , 100 μL of 25‐μM INCA‐6 , or 100 μL of 10‐μg/mL neutralizing antihuman CTGF antibody  were s.c. injected into the parietal bone area. For the negative control, the same volume of PBS or rabbit IgG (Sigma‐Aldrich) was injected. A compression spring was installed on parietal bones 2 hours after the MENK and INCA‐6 injection, or 6 hours after the CTGF antibody injection. Each injection was given again after surgery. During all of the experiments, mice were carefully monitored and no adverse events were observed.After application of the 6‐hour compressive force, mice were perfusion fixed with 4% paraformaldehyde  under inhalation anesthesia with isoflurane. Calvariae, including the parietal bones, were dissected and immersed in 4% PFA at 4°C overnight. The specimens were decalcified with 20% ethylene‐diaminetetraacetic acid (pH 7.4) at 4°C for 7 days. The specimens were then embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin (Sigma‐Aldrich) and eosin .https://imagej.nih.gov/ij/); thresholds of 40 and 255 were used in red channel images. We determined the thresholds by superimposing binary images on the corresponding fluorescent images and confirming overlaps of the signals on the binary images and on the fluorescent images, according to our previous report. In our analysis of nuclear translocation of NFAT, fluorescent NFAT signals that contained nuclei of osteocytes were considered positive.Deparaffinized sections were immersed in 0.01 M citrate solution and microwaved for 2 min, then incubated at room temperature (RT) for 30 min. The sections were treated with 10% donkey serum (Sigma‐Aldrich) in PBS at RT for 1 hour and incubated with primary antibodies against MENK , DOR , NFATc1 , or NFATc3  at RT for 2 hours or at 4°C overnight. In the negative controls, the primary antibodies were replaced by rabbit IgG (Sigma‐Aldrich). After washing with 0.3% TritonX‐100  in PBS, the sections were incubated with donkey antirabbit‐IgG Alexa Fluor 568  at RT for 1 hour. 4,6‐diamidino‐2‐phenylindole  was used for nuclei detection. Fluorescent signals were visualized using a confocal laser scanning microscope system . To quantify the fluorescence‐positive osteocytes, the fluorescent images were analyzed using ImageJ software  in Can Get Signal Immunostain Solution B . After incubation with a peroxidase‐conjugated secondary antibody  at RT for 30 min, the signals were visualized with 3,3‐diaminobenzi‐dine tetrahydrochloride . The images obtained from caspase‐3 immunohistochemistry were analyzed using ImageJ (NIH); thresholds of 0 and 200 were used to quantify the caspase‐3‐positive osteocytes.For the detection of active caspase‐3, deparaffinized sections were treated with 3% H2O2 for 5 min. Peroxidase‐conjugated antibody solution was pipetted onto the sections at 37°C for 10 min. Apoptotic nuclei were then visualized by DAB (Nichirei) at RT for 8 min. Hematoxylin was used for counterstaining. The images obtained from TUNEL staining were analyzed using ImageJ imaging software (NIH). Thresholds of 0 and 160 were used to quantify the TUNEL‐positive osteocytes. TUNEL signals overlapping with nuclei of osteocytes were considered positive.Apoptotic cell death was detected using the Apoptosis in situ Detection Kit  according to the manufacturer's protocol. Deparaffinized sections were incubated in prewarmed protease solution at 37°C for 10 min. For labeling 3′terminals of DNA, TdT reaction solution was pipetted onto the sections and incubated at RT for 2 hours. Endogenous peroxidase was inactivated using 3% HFive lines, which divide the thickness of the parietal bones adjacent to the sagittal suture into six equal parts, were drawn from the dorsal to the ventral side on images of stained sections. The four square fields (30 × 30 μm) on the first and fifth lines, in contact with edges of the right and left parietal bones adjacent to the sagittal suture, were set‐up as the ROI to quantitatively evaluate TUNEL‐positive osteocytes, expression of MENK, DOR, and caspase‐3 in osteocytes, and nuclear translocation of NFATc1 and NFATc3 in osteocytes  supplemented with 5% fetal bovine serum , 5% bovine serum , 100‐U/mL penicillin, and 100‐μg/mL streptomycin (Thermo Fisher Scientific) on culture plates coated with 0.3‐mg/mL type I collagen . For a MENK administration experiment, cells were seeded onto coated 12‐well plates at 1.4 × 104 cells/cm2 on silicone chambers  coated with 0.01% poly‐D‐lysine , and 0.3‐mg/mL type I collagen and cultured for 2 days. Compressive force was generated by 18% constriction of the chambers using a Strex cell stretch system (Strex); it was loaded onto MLO‐Y4 cells for 6 hours. In the control groups, MLO‐Y4 cells were cultured on the stretch chambers without compressive force loading. Cells were harvested to isolate total RNA using RNeasy Kit .To load compressive force onto MLO‐Y4 cells, the cells were seeded at a density of 1.4 × 10The mouse NFATc1 open‐reading frame was amplified by PCR and cloned into the XhoI and XbaI sites of the pLVSIN‐IRES‐ZsGreen1 lentiviral vector . Viral particles were produced in 293 T Lenti‐X cell line  using Lentiviral High Titer Packaging Mix (Takara Bio) following the manufacturer's instructions, and titered using Lenti‐X qRT‐PCR Titration Kit (Clontech). Mice were sacrificed by cervical dislocation; the parietal bones were quickly dissected. After removing the periosteum and the dura mater, 0.5 mm of parietal bones on either side were isolated and minced with scissors. The specimens were immersed in TRIzol Reagent (Thermo Fisher Scientific) and centrifuged. Total mRNA was then isolated using the RNeasy Kit.RNA isolation from the mouse parietal bones was performed according to our previous report.36 MiccDNA was synthesized from 0.4 μg of total mRNA using a PrimeScript RT Reagent Kit (Takara Bio). Real‐time PCR was performed using a Thermal Cycler Dice Real Time System (Takara Bio). The reaction volume was 25 μL, which contained 2 μL of cDNA, 12.5 μL of TB Green Premix Ex Taq II (Takara Bio), and 0.4 μM of sense and antisense primers. The primer sequences are listed in Table Whole‐cell proteins from MLO‐Y4 cells were prepared using Cell Lytic M Lysis Reagent (Sigma‐Aldrich) containing Protease Inhibitor Cocktail (Sigma‐Aldrich). The protein samples were boiled in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride membranes. The membranes were treated with blocking buffer and incubated overnight at 4°C with rabbit antihuman cleaved caspase‐3  or mouse antihuman glyceraldehyde‐3‐phosphate dehydrogenase  antibodies. The membranes were then incubated with horseradish peroxidase‐conjugated secondary antibodies, and developed with a chemiluminescence detection reagent. Chemiluminescent signals were acquired using the Fusion FX Imaging System .2, 1.8‐mM CaCl2, 13.8‐mM glucose, and pH 7.4) containing 5‐μM Fura‐2 AM (Thermo Fisher Scientific), 1.25‐mM probenecid (Thermo Fisher Scientific), and 0.04% Pluronic F‐127 (Thermo Fisher Scientific) for 30 min at 37°C. Fluorescent images were captured with an Olympus IX71  and Hamamatsu EM‐CCD digital camera  at the rate of 1 frame every 5 s for 5 min. One min after image capturing, 100‐nM ionomycin (Wako) was added to the recording buffer. The ratio of fluorescence at wavelengths of 340 and 380 nm were measured in 10 randomly selected cells in each dish, and the average was considered the fluorescence intensity of the sample. The fluorescence intensity in the cells just before ionomycin treatment was regarded as 1. The relative increase in intracellular calcium was assessed using Meta Fluor Software .MLO‐Y4 cells were cultured on coated 3.5‐cm glass bottom dishes for 24 hours, and MENK was administered into the culture for an additional 24 hours. The cells were loaded with recording buffer (200‐mM HEPES, 115‐mM NaCl, 5.4‐mM KCl, 0.8‐mM MgClt test to compare differences between two groups. For more than two groups, we used an ANOVA, followed by the Tukey–Kramer test. p Values less than 0.05 were considered statistically significant.All data are presented as mean ± SD. Data were statistically analyzed by Student's Compressive force of 0.2 N loading onto the mouse parietal bones for 6 hours narrowed the suture width between the edges of the right and left parietal bones adjacent to the sagittal suture (Fig.  To examine whether the increased CTGF influences the expression of MENK and DOR in the compressive force‐loaded osteocytes, we applied a neutralizing CTGF antibody to the parietal bones prior to compressive force loading. No obvious histological change was observed by administration of the neutralizing CTGF antibody in both the nonloaded and the loaded groups (Fig. We previously reported that expression of CTGF in osteocytes is upregulated by a compressive force of 0.2 N loading for 6 hours in the same compressive force‐loaded mouse model as in the present study.13 To  To evaluate the effect of MENK on the osteocyte apoptosis induced by compressive force, exogenous MENK was s.c. injected into the parietal bone area prior to compressive force loading. There was no obvious histological change caused by MENK administration in the nonloaded and the loaded groups (Fig. Our previous study showed that the increased CTGF by compressive force loading induces osteocyte apoptosis in mouse parietal bones.13 To ups Fig. A. TUNELups Fig. B,C. In ups Fig. B,C. On ups Fig. B,C. Theups Fig. D,E. In ups Fig. D,E. Theups Fig. D,E. Theups Fig. D.We next examined expression of NFATs in the mouse parietal bones and MLO‐Y4 cells. Fig. To examine whether the nuclear translocation of NFATc1 induced by compressive force loading stimulates osteocyte apoptosis; INCA‐6, an inhibitor of nuclear translocation of NFATs, was applied to the parietal bones prior to compressive force loading. No obvious histological changes were observed after INCA‐6 administration in the nonloaded and the loaded groups (Fig. Next, we examined whether MENK regulates nuclear translocation of NFATc1 and NFATc3. In the nonloaded groups, ratios of NFATc1‐positive nuclei without and with MENK were 26.7 ± 4.9% and 30.6 ± 6.6%, respectively (Fig. 2+signaling. Real‐time Ca2+ imaging using the Ca2+ indicator dye Fura‐2 AM showed that a Ca2+ ionophore ionomycin elevated the fluorescence intensity of intercellular Ca2+ in MLO‐Y4 cells without MENK treatment (Fig. 2+ signaling mediators, calcineurin A, calcineurin B, and calmodulin in MLO‐Y4 cells were assessed. Expression levels of these mRNAs were significantly decreased by MENK treatment in a dose‐dependent manner (Fig. To further investigate the mechanisms by which MENK regulates nuclear translocation of NFATc1, we focused on Caent Fig. E,F. Theent Fig. E,F. Morner Fig. G. It is generally thought that neuropeptides are produced by nerve cells, but recent studies indicate that bone cells are also producers of neuropeptides. For instance, neuropeptide Y is produced by osteocytes and facilitates bone remodeling. In the present study, we demonstrated, for the first time, that osteocytes produce an endogenous opioid MENK in the mouse parietal bones. In addition, we found the expression of DOR, a major receptor of MENK, in osteocytes. Osteocytes reside in lacunae within the mineralized bone matrix and have their dendritic processes through tiny tunnels called canaliculi, forming the osteocyte lacunocanalicular network via gap junctions. These findings suggest that MENK functions in an autocrine and/or paracrine manner in the osteocyte lacunocanalicular network.It is apparent that neuropeptides play important roles in bone metabolism, and the identification of neuropeptides that promote bone remodeling is a critical issue in bone biology., 15, 16nctions., 38, 39 CTGF is known as a mechanoresponsive factor; its expression is upregulated by mechanical stress. It binds to candidate receptors, such as integrin, lipoprotein receptor‐related protein‐1, and tyrosine kinase receptor A; its signaling facilitates various biological processes, including proliferation, differentiation, and substrate production. We previously reported that increased expression of CTGF in osteocytes by compressive force induces osteocyte apoptosis in vivo and in vitro. In the present study, we loaded compressive force to the mouse parietal bones, resulting in a reduction of MENK expression in osteocytes. A neutralizing CTGF antibody inhibited the compressive force‐induced reduction of MENK. Furthermore, we showed that an increase in osteocyte apoptosis in the compressive force‐loaded parietal bones was inhibited by MENK administration. These results suggest that osteocyte apoptosis in response to compressive force is induced through suppression of MENK expression by CTGF signaling. Osteocyte apoptosis is regulated positively by glucocorticoids and oxidative stress‐related factors and negatively regulated by PTH, estrogen, and androgen. In addition to these factors, neuropeptides, including MENK, can be considered novel regulators of osteocyte apoptosis according to the present study. Aging, loss of mechanical stress under a condition of weightlessness, and glucocorticoid‐induced bone disease cause excessive osteocyte apoptosis and subsequent bone loss. Our finding about the inhibitory effect of MENK on osteocyte apoptosis may contribute to the development of treatments for abnormal bone loss caused by osteocyte apoptosis.Osteocytes are key players of mechanotransduction in bones; that is, they convert external mechanical stress into biochemical signaling., 38 CTG stress., 13 It duction., 42, 43n vitro., 13 In ne loss., 46, 47Because NFAT has a close relationship with apoptosis in various types of cells, we examined the role of NFAT in compressive force‐induced osteocyte apoptosis. Our data showed that NFATc1 and NFATc3 mRNA were highly expressed in bone tissues and osteocytes. Nuclear translocation of NFATc1 in the osteocytes in the parietal bones was enhanced by compressive force, whereas that of NFATc3 was not. These results indicated that translocation of NFATc1, among NFATs, into nuclei was increased in response to the compressive force in osteocytes. INCA‐6, which inhibits NFAT translocation into nuclei, suppressed the increase in osteocyte apoptosis in the compressive force‐loaded parietal bones. Moreover, overexpression of NFATc1 induced expression of apoptosis‐related genes and cleaved caspase‐3 protein in MLO‐Y4 cells. Our findings suggest that osteocyte apoptosis induced by compressive force is advanced through induction of NFATc1 nuclear translocation.2+ signaling pathway is a major mechanism of nuclear translocation of NFAT. That is, Ca2+ influx into cytoplasm leads to calmodulin‐dependent activation of calcineurin, followed by NFAT dephosphorylation and nuclear translocation. Importantly, the Ca2+/calcineurin/NFAT pathway promotes apoptosis in various cells such as muscle cells, megakaryocytes, and mesangial cells. In the present study, we found that MENK suppressed Ca2+ influx and expression of calcineurin and calmodulin in osteocytes, indicating that MENK is a negative regulator of Ca2+ signaling. Taken together, MENK would negatively regulate osteocyte apoptosis through the suppression of the Ca2+/calcineurin/NFATc1 pathway.Furthermore, we analyzed the relationship between MENK and NFATc1 in the molecular regulation of osteocytes apoptosis. Our data showed that MENK administration reduced nuclear translocation of NFATc1 in osteocytes in vivo, suggesting that the decreased MENK expression promotes nuclear translocation of NFATc1 and following osteocyte apoptosis during osteocyte response to compressive force. It is known that the Calocation.48 Impl cells., 49, 502+ influx is an initial biochemical event of mechanical stress‐loaded osteocytes; it stimulates osteocyte functions, such as gene expression and intercellular communication. Therefore, inhibition of Ca2+ influx by MENK may be an important mechanism to regulate mechanical stress‐induced bone remodeling by regulating not only apoptosis, but other functions of osteocytes.Canication.51 The2+ signaling in osteocytes. These results suggest that osteocyte‐produced MENK is a competent neuropeptide that regulates bone remodeling driven by mechanical stress. Our findings about novel roles of MENK in osteocytes not only contribute to understanding the mechanisms of bone remodeling regulated by neuropeptides, but are also possibly applicable to the development of treatments for pathological bone loss.The present study revealed that osteocytes expressed MENK, whereas the MENK expression was suppressed by compressive force via CTGF signaling. Moreover, MENK inhibited compressive force‐induced osteocyte apoptosis by downregulating nuclear translocation of NFATc1. MENK is considered to regulate nuclear translocation of NFATc1 by suppressing CaAll authors state that they have no conflicts of interest.


Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant clonal plasma cell disorder, with a 1% yearly risk of progression to multiple myeloma (MM). Evolution of M‐spike and serum free light chain (sFLC) during follow‐up could identify patients at high risk of progression. In this region‐wide study, including 4756 individuals, 987 patients with MGUS were identified, and baseline factors as well as evolving involved FLC (iFLC) were evaluated as potential markers for risk of progression from MGUS to MM. Furthermore, evolving iFLC and M‐spike were assessed quarterly for a median of 5 years. At baseline, patients that progressed had significantly higher iFLC compared to non‐progressors. The risk factors of M‐spike >1.5 g/dL, age >65 years and iFLC >100 mg/L were all independently associated with increased risk of MGUS to MM progression. For patients that had any two or three risk factors, the 5‐year cumulative probability of progression was significantly higher (31%) compared to no risk factors (2%). Evolving iFLC >100 mg/L during follow‐up was consistently associated with increased risk of progression. Based on our observations, we propose to include iFLC as a monitoring tool for all MGUS patients. Furthermore, we recommend a quarterly monitoring in all high‐risk patients. Finally, we suggest that the risk of MGUS progression should be stratified with age, M‐spike, and iFLC at baseline. It is observed in 3.2% of those aged >50 yearsMonoclonal gammopathy of undetermined significance often precedes MM with a constant annual risk, thus, life‐long follow‐up is necessary.Abnormal FLCr are detected in more than 95% of MM but only in 30%‐47% of MGUS cases.It is of importance to identify high‐risk MGUS groups, both for better risk classification at diagnosis in conjunction with a more optimal monitoring during follow‐up. Furthermore, high‐risk MGUS patients may potentially be considered for preventive measures to limit the progression to MM. As the percentage of patients with detectable iFLC are, and becoming higher in MM compared to MGUS, we hypothesized that evolving iFLC during monitoring may be an essential risk factor for progression. We therefore analyzed a unique dataset from hematology clinics and primary care units in Sweden, with up to 46 years of follow‐up.22.1The study proposal was approved by the Swedish Ethical Review Authority (EPN: 2017/349‐31) and was performed in accordance with the Helsinki declaration.For this study, we queried the Karolinska University Laboratories database, which constitutes data from 20% of the Swedish population, to identify individuals with sFLC measurements from 1 September 2009, until 1 September 2017. All individuals older than 18 years, with an analysis of serum or urine PEP (s/uPEP) within 7 days of sFLC measurement were included. In total, 4756 individuals were included, which generated 30 052 sampling occasions from all individuals during the study period. The electronic medical records for all patients included in the study were reviewed for records of MGUS and MM diagnosis date as well as to be able to exclude patients with other plasma cell disorders or hematological disorders. So, 2003 individuals that had no plasma cell disorders or evidence of M‐protein in s/uPEP or abnormal FLCr were excluded. And, 1766 patients were excluded due to diagnoses of either plasma cell disorders or hematological diseases other than MGUS. Upon further exclusion of the IgM MGUS population, the remaining study population consisted of 987 patients with an MGUS diagnosis of either IgG, IgA, IgD or light‐chain type . All MGUFor each individual, besides sFLC, routine laboratory and clinical parameters were collected and compiled from all available sampling occasions during the study period Table .Regarding s/uPEP; M‐protein of heavy and light chain types, M‐protein size, total immunoglobulin levels, urine kappa and lambda levels, were included in the database. Serum FLC assays were conducted with latex‐enhanced immunonephelometric assay . Total serum immunoglobulin  concentrations were analyzed using immunoturbidimetric assay . Urine light chains (kappa and lambda) concentrations were analyzed using immunonephelometric assay . S/uPEP and immunofixation were performed with agarose gels on the Hydrasys/Hydrasys 2 platform .Sequential data were evaluated in a subgroup of the main MGUS cohort, where at least two serial samples, for example, a baseline sample and one additional sample, with a minimum of 3 months between, per patient were available during the study period and before the progression to MM, and where the sample nearest to MM diagnosis was stipulated to be a minimum of 6 months prior to the date of the diagnosis. The sequential cohort (n = 516), comprised a total of 5364 sampling occasions for all MGUS patients, median serial samples four (range 2‐56). In total, 141 patients had the minimum of two serial samples and 375 patients had three or more serial samples. In patients progressing from MGUS to MM, serial samples before the MM diagnosis were selected, resulting in 44 patients (9%) with MGUS developing MM during the follow‐up time.2.2The endpoint was time from first sample available at MGUS diagnosis (baseline sample) to progression of MM, either smoldering or treatment demanding MM as defined by IMWG.P value threshold <.05) were reported. Log‐rank tests were performed for inter‐group comparisons. The Kaplan‐Meier method was used to calculate medians at 60 months and their associated 95% confidence intervals.The effects of prognostic factors were estimated by univariate Cox regression, where the hazard ratios (HR) (CI threshold: 95% and P < .05) in the univariate regressions using a backward selection, were assessed with Cox proportional hazard regression. Similar to the univariate regressions, the time to event is defined as the time from the first sample available at MGUS diagnosis to the end of follow‐up or progression to MM. Both univariate and multivariate analysis were performed with continuous values to evaluate the effect of subsequent increasing values, where the log value was taken of the plasma/urine M‐spike and the iFLC to better handle extreme values, as well as categorical variables to evaluate the effect of the cut‐off values for risk factors.Multivariate analyses, including all variables significant  of 0.5 g/dL and 100 mg/L were chosen according to the IMWG MM progression criteria.For the selected indicators, time threshold and prognostic variables receiver operating characteristic (ROC) method was used to evaluate the prediction reliability, specificity and sensitivity.33.1The main MGUS cohort included 987 MGUS patients, 530 (54%) were male, and the median age was 68 years (range 26‐95 years). The median follow‐up time was 64 months, 5824 person‐years. There were 904 (92%) patients with MGUS that did not progress to MM (MGUS‐NP), while 83 (8%) patients with MGUS developed MM (MGUS‐MM) during the follow‐up time Table . A sub‐aP < .001). Note, M‐spike >1.5 g/dL at baseline was more frequent in MGUS‐MM  compared to MGUS‐NP . No significant differences between MGUS‐MM and MGUS‐NP in heavy chain types were observed  with available bone marrow assessment compared to 20 out of 78 patients (26%) in patients with M‐spike >1.5 g/dL without available bone marrow assessment. In the main cohort, baseline values of abnormal FLCr were observed in 56 (68%) MGUS‐MM patients compared to 339 (38%) MGUS‐NP (P < .001) compared to MGUS‐NP (P < .001). However, no differences were observed for heavy chain type between MGUS‐MM and MGUS‐NP  and serum M‐spike >1.5 g/dL (P < .001) were associated with increased risk of progression to MM, whereas non‐IgG was not associated with any increased risk.Previously reported factors associated with progression from MGUS to MM are shown in Table 3.4P = .004). In patients with the age >65 years at MGUS diagnosis, the risk of progression to MM was higher compared to patients <65 years of age, , and  for patients >70 vs <70 years.Age was a major correlate to progression, both as a continuous variable and with cut‐offs of >65 years and >70 years. The yearly HR for age as a continuous variable was 1.03 . Furthermore, iFLC >100 mg/L at baseline strongly correlated to MM progression, . As an abnormal FLCr can be anticipated when iFLC levels exceed 100 mg/L, a sub analysis of FLCr as a risk factor in patients with iFLC <100 mg/L was performed. A significant increased risk of progression was observed also in these patients, HR: 2.27, 95% CI: 1.34‐3.85, P = .002.The iFLC concentration correlated with increased risk of progression to MM  while no significance was observed for either age >65 or, interestingly, FLCr. The lack of significance in the later factors is likely affected by the low numbers of patients with LCMGUS.A separate univariate analysis of the LCMGUS cohort including the risk factors  showed an increased risk of progression in patients with iFLC >100 mg/L . For categorical multivariate analysis, age >65 and iFLC >100 mg/L remained significant. While no significance was observed for M‐spike as a continuous variable, when investigating it with the cut‐off reported by IMWG, >1.5 g/dL, the factor was significant, P < .001  concentrations  as group two, thus excluding FLCr for patients with iFLC >100. This combined parameter was entered into multivariate analysis together with age >65 years and M‐spike >1.5 g/dL. The HR for iFLC >100 mg/L, regardless of FLCr, for this model was higher compared to the model including M‐spike >1.5 g/dL, age >65, FLCr abnormal and >100 mg/L Table .P = .36). Therefore, we cannot deduce that hemoglobin is a risk factor for this cohort.Another factor that was significant in univariate/multivariate analyses was hemoglobin. However, when investigating the differences between MGUS‐NP and MGUS‐MM using a chi‐square test Table , no sign3.6To further investigate the impact of the proposed risk factors, M‐spike >1.5 g/dL, age >65 years, and iFLC >100 mg/L, we stratified the patients according to the risk factors. As there was no significant difference, and the TTP‐KM curves were superimposed, between the groups with two and three risk factors, potentially due to the low number of patients with all the three risk factors , these groups were merged. Patients were assessed as low risk , intermediate‐risk , and high risk . For each risk factor added, there was a clear and significant increase in the risk of progression. The five‐year cumulative probability of progression from MGUS to MM significantly correlated with the increasing number of risk factors, 2%, 11%, and 31% for low, intermediate, and high risk, respectively . Evolving M‐spike and iFLC was observed in only two of the eight patients before progression to MM.3.7To explore whether an increase in heavy or light chain concentration during follow‐up, was associated with an increased risk of progression, further analyses were performed in patients with at least two sequential samples, 472 patients with MGUS‐NP and 44 patients with MGUS‐MM.An increase in M‐spike of >0.5 g/dL from baseline value was only significantly associated with increased risk of developing MM at three time periods during the first 5 years; at 9, 15 to 21 months and 30 months Figure . For theAn increase in iFLC of >100 mg/L, from baseline value, was consistently associated with a significantly increased risk of developing MM, 6 months from baseline, and up until 6.25 years, Figure . An incrTo assess the performance of the evolving biomarkers as risk factors, we compared the area under the curve (AUC), sensitivity and specificity for the four factors . Increases in iFLC from baseline was the best predictor for risk of progression .4In this study, we selected 4576 individuals out of which 987 were MGUS patients, with a median follow‐up of 64 months. Patients with MM, AL amyloidosis or other hematological malignancies were excluded. Since the progression of IgM MGUS in our material was exclusively to lymphoma or Waldenstrom's, all IgM MGUS patients were also excluded.The large number of observations available for the sequential cohort enables us to evaluate the real‐world evolution of iFLC. However, the retrospective nature of the present study imposes limitations. As sFLC analyses were started in 2009 in Stockholm, almost half of the MGUS‐MM patients had no available results from before MM diagnoses and were therefore excluded. The lack of bone marrow examinations also limited our ability to evaluate other risk factors such as cytogenetics and flow cytometry.Various models to assess the risk of MGUS progression have been tested, with the Mayo Clinic model being incorporated into the current IMWG guidelines.P = .002). Similar findings were previously reported.Beside the already established risk factors recommended by the IMWG, other groups have suggested that immunoparesis,Knowing that the incidence of abnormal FLCr is more common in MM (>95%) compared to MGUS (30‐42%),Abnormal FLCr can be observed already at low tumor burden with low production of M‐protein. Thus, abnormal FLCr could potentially be observed in a large proportion of the MGUS populations; we observed in our study that abnormal FLCr was present at diagnosis in 397 (40%) patients, which is similar to the 33%‐47% observed in the previous studies.Patients with low risk (30%) had a very low five‐year risk to progression (2%). Only eight (3%) of the patients without any risk factor progressed during follow‐up in our cohort. Thus, we suggest that patients with low risk of progression could be emitted from follow‐up. The five‐year risk of progression observed in intermediate‐risk was 11% compared to 31% in high‐risk patients. On the contrary to low‐risk patients, the increased risk observed in both intermediate and high‐risk patients supports these patients’ regular monitoring. As evolving iFLC seems to be a significant factor during follow‐up, we suggest every 6‐12 months, for intermediate‐risk, and quarterly, for high risk, monitoring with iFLC during the first 5 years.The main goals of this study were to incorporate the iFLC into prognostic models and the monitoring of MGUS patients. The presence of evolving iFLC is a clinically significant risk factor of MGUS progression. Based on the data reported here we propose to monitor iFLC for patients with evolving iFLC values >100 mg/L quarterly. We further recommend that incorporation of M‐spike >1.5 g/dL, age >65 and iFLC >100 mg/L as risk factors in prediction models.Charlotte Gran and Hareth Nahi conceived the study and oversaw overall direction and planning. Charlotte Gran and Hareth Nahi wrote the manuscript with input from all authors. Johan Liwing, Andre Verhoek and Ana Gezin analyzed the data. Evren Alici and Hareth Nahi supervised the project. All authors critically revised the manuscript and approved the final version.Appendix S1: Supporting InformationClick here for additional data file.


Cytomegalovirus (CMV) infection has a significant negative impact on liver transplant (LT) recipients. We aimed to evaluate the efficacy of real‐time DNA quantitative polymerase chain reaction (qPCR) in the early detection of CMV and predicting post‐transplant outcomes.This was a retrospective study that enrolled a total of 49 adult LT recipients between December 2016 and October 2019. Serial CMV qPCR were tested weekly. We used operating characteristic curve analysis to quantify qPCR replication numbers to decide the optimal threshold to predict posttransplant complications and overall survival.p < .05). In the univariate logistic regression model, persistent DNAemia ≥ 4 weeks after anti‐CMV treatment, coexisted bacterial and/or fungal infection, and high CMV qPCR ≥ 180 copies/ml with p < .100. High CMV qPCR ≥ 180 copies/ml  remained to be the only independent risk factors for major complication by the multivariate analysis. The overall 2‐year OS rates were 92.5% and 66.7% in the low and the high qPCR group, respectively (p = .030).The optimal cut‐off value of 180 copies/ml (=164 IU/ml) was determined. We had 40 patients in the low qPCR group (<180 copies/ml) and nine patients in the high qPCR group (≥180 copies/ml). Higher qPCR was associated with more severe CMV disease, early allograft dysfunction, major posttransplant complications, longer ICU stays, and lower 2‐year overall survival  not only indicates a hazard in developing major posttransplant complications but also associates with prolonged and refractory treatment courses. We described that high DNA replication (164 IU/ml) is an indicator of developing major postliver transplant complications. Implementing preemptive treatment for this high‐risk group is mandatory to improve outcomes after liver transplantation. ALTalanine aminotransferaseASTaspartate aminotransferaseAUROCarea under the receiver operating characteristic curveCIconfidence intervalCMVcytomegalovirusGRWRgraft‐recipient weight ratioHRhazard ratioICUintensive care unitIUInternational UnitsLTliver transplantationMELDmodel for end‐stage liver diseaseOSoverall survivalqPCRquantitative polymerase chain reaction1Cytomegalovirus (CMV) is the most common single virus that causes complications in transplant recipients, and influences the outcomes and survival especially during the first few months after transplantation.Current treatment strategies such as prophylaxis or pre‐emptive therapy for liver transplantation (LT) patients can be initiated and guided based on the detection of CMV.Currently, CMV qPCR is the preferred and widely used CMV detection method in transplant recipients due to its high sensitivity, simplicity, and quickness.22.1This study was a retrospective analysis of collected data. A total of 49 adult cases received LT in Chang Gung Memorial Hospital at LinKou, between December 2016 and October 2019, were tested for serial pp65 antigen assays and CMV qPCR for CMV detection during their hospitalization after transplant. All patients satisfied with a standard indication of LT for acute liver failure, end‐stage liver disease, or hepatocellular carcinoma. This study was approved by our Institutional Review Board (No. 202001326B0).2.2Surgical planning and execution were performed in consistency technique as our protocol, and it did not include a convention of splenectomy.2.32.3.12.3.1.1Before transplantation, the serologic tests that detect CMV antibodies (Immunoglobulin [Ig] M and IgG) in donors and recipients were measured by the enzyme‐linked immunosorbent assay. A positive CMV IgG indicates a previous CMV‐infected event. To act in concert with an IgG avidity test, it helped to distinguish a primary CMV infection (high binding strength) from a past episode (low binding strength) in IgM positive patients.2.4To determine whether CMV reactivation was influential for posttransplant outcome, we routinely checked CMV antigenemia assay and CMV qPCR weekly since the surgery until the time the patient was discharged or dead.2.5TM Kit CMV code 52206 Immunofluorescence Assay (Bio‐Rad). Monoclonal antibodies identify CMV pp65 antigen and could be visualized with a fluorescent secondary antibody. A positive antigenemia was defined as at least one antigen‐positive cell per 500,000 peripheral blood leukocytes displaying characteristic fluorescence.Ethylenediaminetetraacetic acid‐treated whole blood samples were collected simultaneously for the antigen assay and the CMV qPCR study. The antigenemia assay was conducted within 6 h of blood sample collection, using the MonoFluoThe CMV DNA was isolated and quantified using the COBAS® AmpliPrep/COBAS TaqMan® CMV Test  according to the manufacturer's instructions. The lower limit of quantitation of test is 137 International Units (IU)/ml. The conversion factor between CMV DNA copies/ml and IU/ml is 0.91 IU/copy  in this assay. A viremia was defined as a positive qPCR, that is, detectable CMV DNA in the blood, even if calculated IU/ml were below the lower limit of quantitation of test.2.63/μl), leukopenia , and/or atypical lymphocytosis (>5%).CMV DNAemia was determined as the detectable CMV antigen and/or CMV DNA in the patient's blood in the absence of clinical symptom or sign.2.7The initiation of anti‐CMV treatment started once CMV DNAemia was detected by either intravenous ganciclovir 5 mg/kg twice per day, or oral valganciclovir 900 mg per day, and the treatment stopped when CMV DNAemia vanished.2.8Instead of routine liver graft biopsy, acute rejection after LT was defined and treated as our previously published article by an elevation of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) two times of normal upper limits in our institution  or a rise greater than 30 IU/L in one day.2.9SD, and numbers with percentages were used for qualitative ones. The quantitative comparison between the two main CMV detect methods was checked using bivariate Pearson correlation. The optimal cut‐off value of DNA replication copy numbers in qPCR to predict major posttransplant complications was determined by the Youden index and its discriminative ability was tested by the area under the receiver operating characteristic curve (AUROC). To compare the categorized variables between the high and the low qPCR groups, Pearson's χ2 test was utilized. In the univariate logistic regression analysis, factors with a p value less than .100 were considered potential ones and further entered into the multivariate model with backward selection. The OS difference was evaluated by the Kaplan–Meier method. Two‐tailed p value was considered to be statistically significant if it was less than .05. All analyses were calculated using SPSS Statistics version 24.0 (SPSS Incorporation).Quantitative parameters were expressed by median values and mean values ±33.1n = 47, 95.9%), and in the majority of cases underwent the surgery with right lobe grafts  with a median GRWR of 0.90% . The leading etiology was hepatitis B virus infection , followed by alcohol use . Among all, 23 (46.9%) patients had hepatocellular carcinoma. With regarding to CMV serologic tests, there were 49 (100.0%) and 41 (83.7%) patients that had positive anti‐CMV IgG positive results in recipients and donors, respectively. In the present study, none of the enrolled patients had a positive IgM result. After the surgery, weekly tests for CMV antigen assay yielded positive results in 22 (44.9%) patients, while 29 (59.2%) patients had detectable CMV DNA (viremia) in their blood. The median viral copies in 1 ml sample were 150 (the mean copy numbers: 231.4 ± 432.6) through CMV qPCR studies. The median length of posttransplant ICU stay was 11 days (the mean: 14.1 ± 8.0 days), and the median hospitalization was 28 days (the mean: 37.3 ± 34.4 days). Four (8.3%) patients had encountered major post‐transplant complications. After a median followed time of 28.5 months (the mean follow‐up: 27.6 ± 9.9 months), six patients died.Demographic data were summarized in Table 3.2r = .755, p < .001). The qualitative results between the two detection methods by week after transplant are shown in Figure The quantitative correlation between the CMV antigenemia and qPCR assays was strong . The maximum copy number ever detected in each patient was used for this analysis. The AUROC of CMV qPCR copies in predicting major complication was 0.764  and higher MELD score  were significantly higher in the high qPCR group. High qPCR levels were associated with detectable antigenemia (p = .028) and persistent DNAemia for 4 weeks after treatment (p < .001) compared to low levels. It exhibited no difference in early detectable DNAemia at postoperative week 1 between the two groups. Regarding clinical outcomes, higher qPCR seemed to be associated with more severe CMV disease, early allograft dysfunction, major posttransplant complication, longer ICU stays, and lower two‐year OS . Besides, a trend to have coexisted bacterial and/or fungal infection was observed in the high qPCR group (p = .072). However, the acute rejection rate distributed equally among the high and low qPCR groups (p = .243).According to the optimal cut‐off value, we had 40 patients in the low qPCR group and nine patients in the high qPCR group. Under posttransplant weekly standardizing CMV surveillance, serial CMV qPCR data were gained for every individual patient. As long as the qPCR level ever exceeded the cut‐off value (180 copies/ml or 164 IU/ml), the patient would be assigned into the high qPCR group. Subsequently, we categorized 40 patients to the low qPCR group and 9 patients to the high qPCR group. Among the nine patients, five of them had persistent qPCR ≥ 180 copies/ml for 3 weeks and another four patients subsided after 1‐week treatment. A comparative study of posttransplanted patients according to high and low qPCR levels was further analyzed, as shown in Table 3.5p < .100. High CMV qPCR ≥ 180 copies/ml  remained to be the only independent risk factors for major complication, as shown by the multivariate analysis.Available clinical factors were analyzed in posttransplanted patients to predict major posttransplant complications, and the results are shown in Table 3.6p = .030; Figure After a median follow‐up period of 28.5 months, the overall 2‐year OS rates were 92.5% and 66.7% in the low and the high qPCR group, respectively. The recipients with higher levels of CMV qPCR demonstrated poorer survival outcomes compared to those who had low levels , but slightly decreased in specificity: 84.4% and 82.2%, respectively. Therefore, 180 copies/ml represents the best discrimination ability of diagnostic decisions. In addition, a higher cut‐off value allows clinicians to have a more flexible space to choose anti‐CMV regimens.The data presented in our study strongly implied that the direct association between high CMV viral loads and adverse posttransplant outcomes, but it had several limitations in addition to the study design . First, the small sample size increased the likelihood of a Type II error. Second, this study was conducted in a single tertiary medical center, and contradiction from protocol bias between other facilities and ours may arise. To this end, the availability of an international standard will improve interlaboratory test result agreement. Third, all recipients in the present study were with positive CMV serologic tests, so a direct effect on disease development should not be ignored. Fourth, this study focused on CMV detection during hospitalization and its impact on posttransplant outcomes, the occurrence of late‐onset CMV disease is out of scope. Therefore, larger and prospective research works are required to confirm our results.In conclusion, our findings support evidence that qPCR is effective and reliable in diagnosing CMV infection and being an early signal to initiate pre‐emptive therapy. Besides, it also provides an objective perspective in predicting post‐transplant care outcomes. High plasma CMV DNA load (defined as CMV qPCR ≥ 180 copies/ml or 164 IU/ml) not only indicates a hazard in developing major posttransplant complications but also associates with prolonged and refractory treatment courses. Implementing preemptive treatment for high‐risk patients is mandatory to improve overall outcomes after LT.The authors declare that there are no conflict of interests.Hao‐Chien Hung and Po‐Jung Hsu participated in the writing of the paper, research design, performance of the research, and data analysis. Jin‐Chiao Lee, Yu‐Chao Wang, Chih‐Hsien Cheng, Tsung‐Han Wu, Ting‐Jung Wu, Hong‐Shiue Chou, and Kun‐Ming Chan participated in research design and performance of the research. Wei‐Chen Lee and Chen‐Fang Lee participated in research design and and data analysis.


Despite a generally high specificity, electrocardiographic (ECG) criteria for the detection of left ventricular hypertrophy (LVH) lack sensitivity, particularly in obesity patients.The aim of the study was to evaluate the accuracy of the most commonly used ECG criteria (Cornell voltage and Sokolow‐Lyon index), the recently introduced Peguero‐Lo Presti criteria and the correction of these criteria by body mass index (BMI) to detect LVH in obesity patients and to propose adjusted ECG criteria with optimal accuracy.The accuracy of the ECG criteria for the detection of LVH was retrospectively tested in a cohort of obesity patients referred for a transthoracic echocardiogram based on clinical grounds . Adjusted ECG criteria with optimal sensitivity for the detection of LVH were developed. Subsequently, the value of these criteria was prospectively tested in an obese population without known cardiovascular disease .2; sensitivity 47% test cohort, 40% validation cohort; for females, the Sokolow‐Lyon index corrected for BMI, (SV1 + RV5/RV6)*BMI ≥885 mm*kg/m2; sensitivity 26% test cohort, 23% validation cohort.Established ECG criteria had a poor sensitivity in obesity patients in both the test cohort and the validation cohort. The adjusted criteria showed improved sensitivity, with optimal values for males using the Cornell voltage corrected for BMI, (RaVL+SV3)*BMI ≥700 mm*kg/mEstablished ECG criteria for the detection of LVH lack sufficient sensitivity in obesity patients. We propose new criteria for the detection of LVH in obesity patients with improved sensitivity, approaching known sensitivity of the most commonly used ECG criteria in lean subjects. Subsequently, the identified optimal criteria were prospectively tested in an obese population without suspicion of or known cardiovascular disease.2The accuracy of the Cornell voltage, Sokolow‐Lyon index, Peguero‐Lo‐Presti criteria, and the correction of Cornell voltage by BMI for detection of LVH was retrospectively tested in obesity patients who were referred for a transthoracic echocardiogram based on clinical grounds (suspicion on or history of cardiovascular disease) (test cohort). From these data, ECG criteria with optimal sensitivity for the detection of LVH were developed by adjusting the cut‐off values and correcting all voltage criteria for BMI. After this, the value of these criteria was prospectively tested in an obese population without suspicion of or history of cardiovascular disease .2.12) who came to the Franciscus Gasthuis and Vlietland  in 2017 and underwent both an ECG and transthoracic echocardiography were included in the analysis. Patients with conditions potentially affecting the ECG voltage amplitude, such as a left or right bundle branch block, a paced rhythm, and imaging evidence of myocardial infarction or pericardial effusion, were excluded.All obesity patients .A standard 12‐lead ECG was recorded at a paper speed of 25 mm/s and an amplification of 10 mm/mV. Heart rate, QRS duration, R‐wave and S‐wave heights, and QRS axis were measured. Left axis deviation was determined as QRS axis between −30° and −90°. Measurements were taken to the nearest 1 mm. The most commonly used ECG criteria were analyzed 3]} + 0.6. LVM was abnormal if LVM ≥225 g for males and ≥163 g for females. The LVM was divided by the body surface area (BSA) to calculate the LVM‐index (LVMI). BSA was calculated by the Mosteller formula.2 for males and ≥88 g/m2 for females.Two‐dimensional (2D) grayscale harmonic images were obtained in the left lateral decubitus position using a commercially available ultrasound system , equipped with a broadband (1‐5 MHz) X5‐1 transducer. All acquisitions and measurements were performed according to current guidelines.2.5t test was used for continuous variables and the χ2 test for categorical variables. Continuous values were expressed as mean ± SD and categorical values as percentages. The sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV), and their 95% confidence intervals were calculated for the ECG criteria in both groups. Differences in sensitivity and specificity between cohorts were tested with the χ2 test. A two‐tailed P value <.05 was considered statistically significant. Optimal cut‐off values for all ECG criteria were manually calculated for both genders by a receiver operating characteristic (ROC) curve, using a fixed specificity of 95%.To compare baseline characteristics between the two cohorts, the Student 33.1A total of 194 patients were included in the test cohort and 100 in the validation cohort. Twenty‐seven patients were excluded in the test cohort; five patients because of a paced rhythm, 15 patients because of a bundle branch block, and seven patients showed wall motion abnormalities on echocardiography. No patients were excluded due to poor echocardiographic windows.2, P < .001) compared to the test cohort. LVM , and LVMI  were not significantly different between groups. However, the prevalence of LVH (defined as an increased LVMI) was higher in the test cohort . LVH criteria as measured by ECG and echocardiography were stratified by gender as well  followed by the Peguero‐Lo Presti criteria . The Sokolow‐Lyon index had very poor sensitivity  in this obese population *BMI ≥700 mm*kg/m2, had the best sensitivity for males , specificity 96% (CI: 78%‐100%), ROC AUC 0.65, PPV 90% (CI: 54%‐99%), and NPV 71% (CI: 52%‐84%). The Sokolow‐Lyon index*BMI, (SV1 + RV5/RV6)*BMI≥885 mm*kg/m2, had the best sensitivity for females , specificity 93% (CI: 85%‐97%), ROC AUC 0.69, PPV 60% (CI: 33%‐83%), and NPV 77% (CI: 68%‐84%).New cut‐off values for both males and females were defined for all criteria, with a fixed 95% specificity. The new cut‐off values for the Cornell voltage, Sokolow‐Lyon index, and Peguero‐Lo Presti criteria were, respectively, ≥20, 24, and 19 mm for females and ≥27, 27, and 23 mm for males. All criteria were multiplied by BMI. The optimal cut‐off values for the Cornell voltage*BMI, Sokolow‐Lyon index*BMI, and Peguero‐Lo Presti criteria*BMI were, respectively, ≥795, 885, and 780 mm*kg/m3.4When the new criteria were tested in the validation cohort, again the adjusted Cornell voltage*BMI had the best sensitivity for males , specificity 92% (CI: 71%‐99%), ROC AUC 0.78, PPV 50% (CI: 9%‐91%), and NPV 88% (CI: 68%‐97%). The Sokolow‐Lyon index*BMI again had the best sensitivity for females , specificity 83% (CI: 70%‐91%), ROC AUC 0.57, PPV 25% (CI: 7‐57%), and NPV 81% (CI: 68%‐90%). None of the male patients in the validation cohort had a positive Sokolow‐Lyon index at a cut‐off value of 27 mm. There were no substantial differences between the sensitivity and specificity in the test cohort and validation cohort.42 for males and (SV1 + RV5/RV6)*BMI≥885 mm*kg/m2 for females, for the detection of LVH in obesity patients with improved sensitivity, without losing specificity.In the current study, we demonstrated that in obesity patients, established ECG criteria for the detection of LVH lack sufficient sensitivity for application in daily clinical practice. We propose new criteria, (RaVL+SV3)*BMI≥700 mm*kg/mThe explanation of the poor sensitivity of the established ECG criteria for detection of LVH  may be that obesity patients commonly have reduced voltages in the precordial ECG leads, probably because the ECG voltages at the skin level are attenuated by the subcutaneous adipose tissue.Because in obesity patients with cardiac disease there may often already be a clinical indication for an echocardiogram, an ECG as a screening tool for detection of LVH may have the most value in obesity patients without known cardiac disease. In the current study for the first time, adjusted ECG criteria for the detection of LVH were tested in such a relatively low‐risk obese population. Even in these subjects, the proposed new criteria performed fairly very well . Although these sensitivity values appear to be rather poor, also in lean subjects the sensitivity of ECG criteria for LVH is known to be limited. A review of multiple studies in different healthcare settings found that the sensitivity of the Cornell voltage and Sokolow‐Lyon index ranged from 2% to 52% with a specificity ranging from 71% to 100%.2, which is much lower than the mean BMI of our test cohort and validation cohort .When adjusting the Cornell voltage by BMI as designed by Angeli et al.,Some other studies regarding the optimization of ECG criteria for the detection of LVH in obesity patients have been performed before. Rider et al. made an adjustment to the cut‐off value of the Sokolow‐Lyon index (+8 mm). This improved the sensitivity to 27% (specificity 93%) in their test cohort and 25% in their validation cohort.P < .001) leading to smaller S wave amplitude in V3, which measures posteriorly directed myocardial electrical activity.All adjusted criteria in our study had better sensitivities in males than in females. This difference is possibly because of the abundant breast tissue in obese women,Finally, it may seem difficult to implement our proposed criteria into daily practice since, apart from the necessity to assess BMI, it would require the use of different criteria in males and females. However, nowadays ECG devices already use programmed algorithms for standard interpretation. It will be a relatively minor issue to add our proposed new criteria to these modern devices, allowing easy clinical use without extra effort.4.12, all patients included in our study had a BMI ≥35 kg/m2 because this was an inclusion criterion for the CARDIOBESE study. Therefore, the conclusions may only be applied to morbidly obese patients and not to obesity patients in general. The sample size was relatively small. The validation cohort had a relatively low prevalence of LVH; therefore, the PPV values even for the new criteria were low. However, as mentioned before, in the current study for the first time, adjusted ECG criteria for the detection of LVH were validated in a relatively low‐risk obese population without known or suspicion of cardiovascular disease. Racial differences in the diagnosis of LVH were not addressed in this study. Also, the abilities of the proposed criteria to predict outcomes  are not known. Finally, we included only BMI as an obesity index and could not assess whether, for example, waist circumference or epicardial fat thickness is superior to BMI to adjust the voltage ECG criteria.LVM was estimated by 2D echocardiography, despite reports demonstrating superior accuracy of cardiac magnetic resonance imaging, especially in obesity patients.52 for males and (SV1 + RV5/RV6)*BMI ≥885 mm*kg/m2 for females, for the detection of LVH in obesity patients with improved sensitivity , approaching known sensitivity of the most commonly used ECG criteria in lean subjects.Established ECG criteria for the detection of LVH lack sufficient sensitivity in obesity patients. We propose new criteria, (RaVL+SV3)*BMI ≥700 mm*kg/mNone declared.


Anolis lizards), Gymnophthalmidae, and Sphaerodactylidae. Data suggest trophic spider–lizard associations between Ctenidae and Dactyloidae, followed by Theraphosidae and Liolaemidae. The body sizes of the spiders and lizards showed a positive relationship, and spiders were smaller than their prey. We conclude that various spider taxa can be considered lizard predators and they may be ecologically important in the Neotropical and Andean regions. However, spiders of prime predation relevance seem to be those of the Ctenidae and Theraphosidae families.Vertebrate predation by invertebrates has been classically underexplored and thus underestimated, despite the fact that many arthropods consume vertebrates. To shed some light on the relevance that spider predation may have upon lizards in the Neotropical and Andean regions, we compiled the available information in the literature on this trophic interaction. We found 50 reports of spiders consuming lizards in these regions, and the 88% of these were from the Neotropical region. Spiders belong to eight families, but Ctenidae and Theraphosidae were the most frequently reported predators. Lizards belong to 12 families, and the most commonly consumed species corresponded to the families Dactyloidae (all  In this study, we analyze for the first time the lizard consumption by spiders in the Neotropical and Andean regions, showing that the families Ctenidae and Theraphosidae may be relevant lizard predators in these areas. In addition, we provide the first report on spider predation upon a vertebrate taxon in the Andean region. In manyAmong arthropods, spiders are considered one of the main vertebrate predators Valdez, . In factThe information on predator–prey interactions between spiders and lizards is scattered, and although some attempts have been made to systematize this information Bauer, , we haveOriginally, the Neotropical region was defined according to the Sclater–Wallace system, to comprise South America and part of Central America, extending northbound as far as central Mexico Sclater, . This sc2We conducted an exhaustive literature review on spiders consuming lizards in the Neotropical and Andean regions. We searched for information using Google Search, Google Scholar, Thomson‐Reuters, and Scopus databases. The keywords searched were spider, Arachnida, lizard, Lacertilia, predation, Neotropical, Andean, and Andes, used in different combinations. The quest was repeated using the same keywords in Spanish and Portuguese. Finally, we searched information throughout the entire collection of Herpetological Review (1967–June 2020). We considered a “predation event” reports on spiders hunting, seizing, and/or consuming a lizard. In addition, we included reports in which a dead lizard, trapped in a spider web or found in close proximity to a spider burrow, showed clear signs of been consumed by a spider, that is, mastication, envenomation, and/or external digestion.We explored relationships between the sizes, activity patterns , and hunting strategies (active forager vs. ambush predator) of predators and prey, obtaining these data from additional publications for all the species reported in our literature search. We searched for the adult sizes, snout–vent length for lizards, and carapace length for spiders, and this information was used for the analyses, because these data were not always available in the reports. For species that exhibited sexual dimorphism, we reported the body sizes of both sexes when possible, although we restricted the analyses to the females’ body sizes. The information on activity patterns and hunting strategies may help to assess the probabilities that predator and prey come in contact with each other  was evaluated with a Pearson correlation, and the body size differences between predator and prey were assessed with a 3Table The geographical distribution of the predation reports is shown in Figure The predator spiders of lizards belong mostly to the suborder Araneomorphae, which includes seven families: Araneidae (orb‐weaver spiders), Ctenidae (wandering spiders), Lycosidae (wolf spiders), Salticidae (jumping spiders), Sicariidae (six‐eyed sicariid spiders), Sparassidae (huntsman spiders), and Trechaleidae . The second suborder represented was Mygalomorphae, for which predation events only included the family Theraphosidae (tarantulas). The two main predator families were Ctenidae and Theraphosidae, and the less represented were the families Sparassidae and Sicariidae Figure .Lizards consumed by spiders belonged to 12 families Table , from whAnolis . There was, however, no association between the activity patterns (p = .51) and hunting strategies (p = .34) of spiders and lizards. The tendencies observed were a higher predation by nocturnal spiders upon diurnal lizards, and that the most predated lizards have ambush predator strategy, which were consumed mainly by ambush spiders, and secondly by active forager spiders , at Cerro Mariposa, Central Chile . We observed an adult of L. lemniscatus being caught by an adult of Grammostola rosea  (Theraphosidae)  Figure . The liz) Figure . SubsequLiolaemus nitidus : This observation was conducted on spring, 21 September 2012 (14:50 hr), during an exploratory trip to Altos de Cantillana, Central Chile . Underneath a stone, we found a juvenile of L. nitidus being eaten by a spider of the genus Euathlus Ausserer, 1875 (Theraphosidae)  Figure i. The sp) Figure i. Approx) Figure ii. The h) Figure ii.Liolaemus tenuis (Duméril & Bibron 1837): The observation was made on summer, 20 February 2018 (˜11:30 hr), at Lo Barnechea, Central Chile . We found the corpse of an adult of L. tenuis out of a burrow dwelt presumably by a tarantula G. rosea   Figure i. This a) Figure , as obse) Figure ii.In addition to the predation events reported in the literature, we describe three new events, for which we did not collect or measure the animals.4This is the first comprehensive review on spiders consuming lizards from the Neotropical and Andean regions. All reports were anecdotal observations and in most cases (89%), observations started when spiders were consuming their prey. Thus, for most cases it is unknown if spiders initiated the predation event, but scavenging has a limited occurrence in wild spiders, and this behavior has been only observed in laboratory settings, under induced conditions  The larger extension of the Neotropical relative to the Andean region provides bigger area to house more species, as predicted by the species–area relationship proposed by Connor and McCoy . (2) TheCtenus. Ctenids consumed mainly lizards of the families Dactyloidae and Gymnophthalmidae, which may result from their high diversity, abundance, and wide distribution in the Neotropical region (Midtgaard, Ctenidae spiders were the most predaceous species, particularly those of the genus G. rosea and Euathlus sp. upon three Liolaemus species. The reported Liolaemus predators are vertebrates (i.e., snakes, lizards, raptors, and mammals; Jaksić et al., Liolaemus predators. Moreover, G. rosea (Aguilera et al., Euathlus spiders (Canals et al., Theraphosids were the second more predaceous spider group after the ctenids, consuming the largest prey. In fact, theraphosids are the largest extant spiders Foelix,  and thusSpiders have a generalist diet (Riechert & Harp, The activity patterns and hunting strategies of predators and prey did not show any significant association, which suggest that at least these two factors are not modulating the predator–prey interactions. Considering however, the trends that nocturnal spiders consumed diurnal lizards, and that ambush lizards were consumed by ambush predators, we propose that a larger sample size may confirm these trends.The body sizes of spider predators and lizard prey showed a significant positive relationship, similarly to the one observed in other interactions with spiders as predators, and invertebrates (e.g., Bartos, The relevance of these spider‐lizard predator–prey interactions prompts for additional efforts to clarify the real impact that spider predation impose upon lizard populations (Nordberg et al., This review has allowed disclosing interaction patterns between spider predators and lizard prey in the Neotropical and Andean regions, where ctenids and theraphosids apparently may be the most relevant spider predators of lizards. Moreover, in some areas such as in the Chacoan subregion along the Atlantic coast, spiders may play a significant role in shape the lizards’ community structure, considering the high frequency of predation reports from this subregion. The identification of these invertebrates as lizard predators prompts for further quantitative studies on the importance of this type of trophic interactions in the communities of these regions. Quantify this impact will provide significant insights to understand the intraguild, and so bidirectional, interactions between spider and lizard populations, and how these modulate the different interactions at the community level. In addition, and highly relevant during the present Anthropocene, the understanding of these trophic relationships may also contribute in the design of conservation strategies for species that are threatened (Leroy et al., The authors declare that they have no conflict of interest.Claudio Reyes‐Olivares: Conceptualization‐Lead, Data curation‐Lead, Formal analysis‐Lead, Investigation‐Lead, Methodology‐Lead, Project administration‐Lead, Resources‐Lead, Software‐Lead, Supervision‐Lead, Validation‐Lead, Visualization‐Lead, Writing‐original draft‐Lead, Writing‐review & editing‐Lead; Andrés Guajardo‐Santibáñez: Conceptualization‐Equal, Data curation‐Equal, Investigation‐Supporting, Validation‐Supporting, Writing‐review & editing‐Supporting; Bernardo Segura: Conceptualization‐Equal, Data curation‐Equal, Investigation‐Supporting, Validation‐Supporting, Writing‐review & editing‐Supporting; Nicolás Zañartu: Conceptualization‐Equal, Data curation‐Equal, Investigation‐Supporting, Validation‐Supporting, Writing‐review & editing‐Supporting; Mario Penna: Project administration‐Supporting, Supervision‐Supporting, Validation‐Supporting, Writing‐review & editing‐Supporting; Antonieta Labra: Conceptualization‐Lead, Data curation‐Lead, Formal analysis‐Lead, Funding acquisition‐Lead, Investigation‐Lead, Methodology‐Lead, Project administration‐Lead, Resources‐Lead, Software‐Lead, Supervision‐Supporting, Validation‐Lead, Visualization‐Lead, Writing‐original draft‐Lead, Writing‐review & editing‐Lead.Table S1Click here for additional data file.


Ketamine is a widely used anesthetic in experimental medicine. We have also used ketamine for surgical interventions and imaging in rats and found significantly impaired ossification between identically performed experiments, which only differed in the number of anesthetic events. In order to investigate this phenomenon, we estimated the absorbed ionizing radiation and also studied whether ketamine administration has disadvantageous effect on bone cell viability.Spongious bone chips and parietal bone disks were harvested from rats. Explants were incubated in stem cell media containing 0.02, 0.2 and 2 mM ketamine. After 3 days of incubation, tetrazolium-based spectrophotometric assay was performed to measure cell viability. Size-specific dose estimation was used to calculate ionizing radiation of computed tomography imaging.We found that ketamine supplementation with 0.2 mM slightly decreased cell viability, while 2 mM caused significant reduction both in the spongious and cortical explants. The cumulative ionizing radiation was found to be negligible compared to irradiation dosages used to impair ossification.We conclude that multiple ketamine administration was responsible for the diminished regenerative potential of bone tissue in the present experimental setup. For this reason, we suggest that ketamine anesthesia should be avoided in studies investigating bone regeneration. Regeneration is a complex process influenced by uncountable factors, for example, anesthesia-related stress, substances or radiation. The present work is based on an observation from previous experiments investigating bone healing , 2. In oTwo experimental studies were performed previously with the same surgical and evaluation methods. Briefly, under ketamine–xylazine anesthesia , bone defects were created on the rat calvarium with a terphine bur . Computed tomography (CT) was performed in order to visualize bone healing. In order to perform CT scans, anesthesia was induced again. Demineralized bone matrix-filled defects were compared on the 11th postoperative week, whereas increasing bone density and decreasing remaining bone defect size indicated bone regeneration.In Experiment 1, until the 11th week, anesthesia was performed 6 times, during surgery and then biweekly . TherefoIn the present experiment, 230–250 g male Wistar rats  were used. The animals were maintained on lab chow and tap water ad libitum in plastic rat cages with 12-h day/night cycle in the animal facility of the Institute of Clinical Experimental Research in Budapest. All procedures performed in studies involving animals were in accordance with the ethical standards of the local Animal Research Committee of Semmelweis University, Budapest, Hungary (Prof. Dr. György Wéber), and the National Scientific Ethical Committee on Animal Experimentation, Budapest, Hungary .All experimental protocols were approved by the local licensing committee of the Institute of Clinical Experimental Research, Semmelweis University, Budapest, Hungary.n = 7/concentration). Ketamine-free medium was used as control (n = 14).Male Wistar rats were euthanized with high concentration of carbon dioxide in a closed plastic chamber. Spongious bone chips of equal size from the femur epicondyle were harvested. Additionally, cortical bone pieces from the parietal bone were cut with a 4-mm internal diameter trephine bur with slow rotational speed and low pressure. Bone pieces were immediately put into pre-heated (37 °C) stem cell culture medium . Bone pieces were separated and cultured under standard cell culture conditions for 3 days . Ketamine  was added to the medium in 0.02 mM, 0.2 mM, and 2 mM concentrations . Briefly, after 3 days of culture, media were removed and fresh media (150 μl) containing 50 μl XTT (1:9) were added . After 4 h of incubation, the absorbance was measured using spectrophotometer at the characteristic wavelength of 492 nm. The background was measured at 650 nm. Absorbance values were normalized with the weight of the bone pieces.Size-specific dose estimation (SSDE) is a known method to calculate absorbed dose of CT scans from the CT dose index (CTDI) and patient size. According to the method provided by the American Association of Physicists in Medicine, a conversion factor can be calculated to estimate radiation dose .The average rat effective diameter from 15 animals (cm) were calculated. CTDI was 45,7 mGy for every scan. For the present study, CTDI values were given using a 16-cm phantom.p < 0.05 were considered significant.All values are reported as mean ± SEM. One-way ANOVA and Dunnett’s multiple comparison and t-tests were performed using GraphPad Prism statistical software. Probability values of 2 remaining bone defect, while animals in Experiment 2 showed full consolidation without remaining bone defects on the 11th postoperative week  showed significantly higher bone density . Non-significant reduction in cell viability was noticed after administering 0.2 mM ketamine (0.50 ± 0.04). Adding 2 mM ketamine to the medium, however, significantly decreased cell viability (0.14 ± 0.04) Fig. .Fig. 2C2. The conversion factor was 1.66 ± 0.001, meaning, that the SSDE equals to approximately 75.86 mGy for each computed tomography scan in the present experiment. For this reason, the cumulative absorbed dosage for those animals, who have had 5 CT scans was 379.3 mGy.The average effective diameter was 3.12 ± 0.2 cmIn the present experiment, we showed that increasing ketamine concentration decreases the number of metabolically active cells on bone explants. This phenomenon could be responsible for the diminished regenerative potential of bone tissue after repeated anesthetic events.Ionizing radiation could also reduce tissue regeneration. In fact, 20–50 Gy irradiation is generally used to study impaired osteogenesis in rats –6. LowerKetamine on the other hand, was shown to be toxic to hepatocytes, urothelium, and neural cell –10. AddiIn conclusion, we found decreased bone cell viability after ketamine supplementation in a dose-dependent way in vitro, which suggests that the substance also influences osteogenic progenitor activity in vivo. Other factors like xylazine, radiation, or stress alone could also influence healing, but according to the present results, we conclude that ketamine had a pivotal role in decreasing osteogenic regeneration in our previous in vivo experiments. For this reason, we suggest that ketamine anesthesia should be avoided in studies investigating bone regeneration.


Micrococcus luteus, P. mirabilis, B. cereus, E. coli, and S. epidermidis were evaluated. In both Kirby–Bauer tests and liquid culture growth inhibition assays, phosvitin inhibited M. luteus, a microorganism that thrives in the human mouth, but not the other bacteria tested. The MIC of phosvitin was determined to be 31.3 μg/mL when delivered in 1 mM CaCl2 but was 0.5 mg/mL in the absence of added calcium. Expanding on the potential impacts of phosvitin on the mouth, its action was evaluated in a model of tooth decay represented by acid-damaged hydroxyapatite discs. SEM, AFM, and FAAS analyses revealed that pretreatment of discs with phosvitin modulated the damage-induced morphology and topography changes associated with acid-damaged discs.Phosvitin, the most highly phosphorylated metal-binding protein found in nature, binds more than 100 calcium ions, and has been identified as an agent that could be used to generate biomineralization scaffolds. Because of published reports describing phosvitin's affinity for calcium and potential antibiotic activity, this study was undertaken in order to evaluate phosvitin for both antibiotic activity against common microorganisms and the ability to protect hydroxyapatite surfaces from acid damage. To more clearly define its antibiotic action, the effects of phosvitin on  Tooth decay affects 45.8% of the US population aging from 12 to 19 years old , 4, 6 an− and C−termini [Phosvitin is the most phosphorylated protein found in nature , 12 and −termini , 19. ForProteus mirabilis can often be found in the gastrointestinal system and can cause urinary tract infections [Bacillus cereus is a Gram-positive toxin-producing bacterium that is common to soils and in foods and can cause infections of the intestinal tract, the eye, and the lungs and of wounds [Escherichia coli is the most abundant aerobic bacterium residing in the human gut, and although several strains are a commensal part of the human microbiota, some strains cause infections [Streptococcus epidermidis is the leading cause of infections due to placement of surgical materials [Micrococcus luteus, a microorganism common to the human skin and mouth [Micrococcus luteus may also be a causal factor in formation of dental caries [Phosvitin antimicrobial activity was tested against a panel of bacteria that were chosen not only for their availability, but also because they are often part of the human biome—some in health and others in disease. fections , 22. Bacf wounds , 24. Escfections , 26. Whifections , the invaterials . And finnd mouth , is not nd mouth , 31. Micl caries .Micrococcus luteus, but not towards other bacteria tested in Kirby–Bauer disc diffusion assays. At the same time, the presence of phosvitin in a calcium phosphate precipitation assay revealed that inclusion of phosvitin increased the amount of precipitate formed. In addition, SEM, AFM, and FAAS analyses of measures of damage to hydroxyapatite discs demonstrated that treatment with phosvitin modulates acid-induced damage to hydroxyapatite.The research presented here indicates that phosvitin harbors antibiotic activity towards Hydroxyapatite discs were purchased from Clarkson Chromatography Products. Phosvitin was a kind gift from Dr. Dong Ahn at Iowa State University and was prepared according to Lee's production method –35. AddiµM. To a new 1.5 mL Eppendorf tube, 500 µL of each solution was added then mixed with 10 µL of both 1.0 M calcium chloride and 0.6 M diammonium phosphate to form hydroxyapatite precipitation in solution. The mixtures were incubated at room temperature for 4 hours to allow for precipitation. Sample were then centrifuged at 10,000 rpm for one minute to form a pellet, whereupon excess supernatant was carefully removed. The pellet was then stained with 500 µL of 5% alizarin red solution in 75% ethanol. Twice, the slurry was vortexed for 30 seconds and centrifuged at 10,000 rpm; the pellet was then washed with a 75% ethanol wash solution to remove any remaining free alizarin red stain. To dissolve the hydroxyapatite, the final resuspended pellet was placed in 30% acetic acid with 100 µL mineral oil to prevent evaporation and heated on a thermal mixer at 85°C for 15 minutes at 300 rpm. Then, 200 µL of each sample was transferred to a 96-well microtiter plate and the absorbance at 405 nm was determined. The percent change in hydroxyapatite formation for a given phosvitin calculation was determined by taking the ratio of the measurement from the phosvitin experiment to that of the control sample that contained no phosvitin. A standard curve was produced by mixing appropriate volumes of calcium and phosphate solutions at a 1:1 ratio in a 50 mM HEPES buffer solution.The Alizarin Red Hydroxyapatite Crystal Assay was based on Microtiter Plate Hydroxyapatite Inhibition Assay by Xiao et al. (2015) ; howeverThe acid-immersion model of tooth decay developed by Lim et al. (2009)  was usedDried hydroxyapatite discs were fixed to a scanning electron microscopy mount with carbon tape. The disc mounts were then placed in a Cressington Sputter Coater 108 Auto and coated with a layer of gold particles for one minute at full voltage. SEM images were acquired with a TESCAN Vega 2 Scanning Electron Microscope at a 20.0 working distance using a secondary electron detector at an accelerating voltage of 20 kV. Imaging of each sample was performed in triplicate; the specific details associated with the recorded images are presented in the figure legends.Acid-treated discs were mounted to AFM metal mounts with carbon tape then placed onto the main stage area of a Bruker Innova Atomic Force Microscope. Each disc was aligned with the AFM laser and cantilever; acquisition settings are reported with the corresponding figure.2O and placed in a 50 mL cylindrical reflux tube, to which 0.4 mL of 1:1 nitric acid and 0.2 mL 1:1 hydrochloric acid was added. The immersion solution was then heated at 100–105°C using a heat block; sample volume was reduced to 10 mL and refluxed at 95°C for 30 minutes. Cooled samples were diluted to 20 mL with ddH2O then passed through a 0.2 µm filter to remove particulates.Our protocol largely followed that provided by the Environmental Protection Agency  and as rµL of lanthanum solution to depress the response from oxyanions such as free phosphates, aluminates, and silicates that are present after the digestion. Calcium concentration was determined against a calcium stock standard set in serial dilution of 1 to 3 ppm. All measurements were performed in triplicate.Just prior to analysis, nitric acid-digested samples (20 mL) were treated with 100 Flame atomic absorption spectroscopy was performed using a Thermo Elemental Solaar S-Series Atomic Absorption Spectrometer operated under acetylene gas (10 psi) mixed with air (30 psi). Additional instrument settings are provided in the relevant figure legends.M. luteus, P. mirabilis, S. marcescens, P. aeruginosa, B. cereus, E. coli, and S. epidermidis was examined using Kirby–Bauer disc diffusion assays [2, and 25 mg/ml phosvitin in 1 mM CaCl−2 were prepared directly from a lyophilized powder. Bacteria were grown in Luria–Bertani (LB) broth at 37°C for 24 hours in shakers at 250 rpm; then a lawn of bacterial solution was spread across the plate. Plates were treated with discs created from autoclaved filter paper that was dipped into the proper phosvitin solution immediately before placement. Lawns were cultivated at 37°C for 48 hours to obtain the zones of inhibition. Discs treated with 1 mM CaCl2 and 1 mM ZnCl2 solutions were used as controls.The antimicrobial activity of phosvitin towards n assays . SolutioM. luteus and B. cereus was determined according to the method of Andrews (2001) [2 to ensure maximal loading of calcium on the protein. A twofold serial dilution was prepared using concentrations of calcium chloride bound phosvitin from 1 mg/ml to 3.9 μg/ml in LB broth with 1 mM CaCl2 LB broth as a control. Bacteria were grown in LB broth at 37°C for 24 hours and 10 μL of bacterial solution was used to inoculate dilutions. Treatments were cultivated at 37°C for 48 hours at 250 rpm on a shaker.The minimum inhibitory concentration (MIC) of phosvitin towards s (2001) . These MM. luteus in solution in the absence of added calcium was also determined, as were the effects of casein, or bovine serum albumin (BSA). To achieve quantitation, turbidity was measured at an absorbance of 600 nm according to the method of Koch (1970) [The ability of phosvitin to impair the growth of colonies of h (1970)  using a To measure the formation of hydroxyapatite, the method of Xiao et al. (2015)  was usedIn general, the alizarin red assay provides a measurement of the extent of calcium phosphate formation as a pellet. However, this assay has weaknesses; subtle changes in pH cause differences in the spectral properties of the dye, and the calcium phosphate precipitate formed does not represent the hydroxyapatite found in enamel. To develop a more reliable assessment, an acid-damage model using hydroxyapatite discs was used; readouts included structural methods including SEM, AFM, and FAAS.Purchased hydroxyapatite discs were damaged in an acid immersion assay, and the influence of phosvitin pretreatment was assessed. The surface of the untreated acid-damaged discs was rough and contained pits and cracks . PhosvitThe surface topography of the acid-damaged hydroxyapatite discs was assessed using AFM . UntreatThe solution that was retained after a hydroxyapatite immersion experiment was subjected to flame atomic absorption spectroscopy (FAAS) in order to quantify the extent of release of calcium ions in solution.R2 of 0.9972. Phosvitin-naïve discs immersed in a pH 3.26 solution for one day released an average of 0.557 ppm calcium ions (n = 6) in solution, whereas after one-month time the data report an average of 4.6 ± 0.2 ppm calcium ions released into solution. However, increasing concentrations of phosvitin up to 1 mg/mL resulted in diminished calcium release in comparison to phosvitin-naïve discs. The concentration of calcium ions released was 0.7 ± 0.1 ppm, 0.5 ± 0.1 ppm, 0.54 ± 0.07 ppm, and 0.3 ± 0.2 ppm when discs were treated with phosvitin at concentrations of 0.13 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL, respectively. Regardless of the concentration of phosvitin tested, incubation of discs with the protein resulted in significant retention of calcium ions in this acid damage assay.Taken together, the SEM, AFM, and FAAS results demonstrate that phosvitin is mitigating damage to hydroxyapatite discs in the acid immersion experiments. SEM data provides a qualitative description of how treatment of discs with phosvitin leads to surface morphology changes that reflect a reduction in cracks compared to damaged discs naïve of phosvitin. The AFM data indicates an overall decrease in surface roughness. The third and final set of data to be taken into consideration is the FAAS data that quantified the extent to which phosvitin impeded the dissolution of calcium ions from hydroxyapatite discs at all concentrations tested.B. cereus was slightly inhibited by 25 mg/ml phosvitin in 1 ZnCl2 and 25 mg/ml phosvitin in 1 mM CaCl2 . Overall, these data indicate that phosvitin robustly inhibited M. luteus when provided in the presence of calcium, but only modestly in the absence of added calcium. Use of bovine serum albumin nor casein reproduced these effects.1 mM CaCl2 represents a strong concentration of calcium ions. Whether the inclusion of 1 mM calcium ions was affecting .5 mg/mL . NeitherE. coli and S. aureus in a model of infection of zebrafish embryos [M. luteus, a common Gram-positive to Gram-variable microorganism of the human microflora [M. luteus. While M. luteus is not considered the main causative agent in dental caries [There is a prior report of phosvitin proving bactericidal towards  embryos , indicatcroflora , expandil caries , it is al caries , 49. An l caries , septic l caries , meningil caries , and pnel caries .M. luteus when delivered with 1 mM CaCl2, having an MIC of 33 mg/mL, a rather potent value. However, this potency is lost in the absence of calcium; furthermore, phosvitin did not inhibit other microorganisms tested to an appreciable extent. The relevance of calcium in phosvitin has been reported in the literature; phosvitin's three-dimensional conformation at air–water interfaces is dependent on calcium [M. luteus.Our data indicates that full-length phosvitin is potent as an antibiotic agent towards  calcium . Indeed, calcium  propose Phosvitin increased calcium formation in a calcium phosphate precipitation assay and mitigated acid damage to hydroxyapatite discs. This is consistent with the data from Belhomme et al. which suThere is also some interest in whether component peptides of phosvitin can bind calcium and/or retain biological activity. While we examined whether thermolysin or pepsin digests of phosvitin demonstrated activity in our assays, the unpurified digests did not stimulate formation of a calcium pellet to the same extent as full-length phosvitin ; neitherM. luteus, but not against other bacteria tested. For these reasons, phosvitin may prove to be of great interest in maintaining the health of the mouth both in terms of protecting hydroxyapatite of teeth against acid damage and against potential cariogenic activity M. luteus, a microorganism common to the mouth.The SEM, AFM, and FAAS analyses reported here indicate that treatment of hydroxyapatite discs with phosvitin protect the discs against acid damage. Furthermore, phosvitin has been shown to possess antimicrobial activity towards