Hypomagnesemia is commonly observed in heart failure, diabetes mellitus, hypertension, and cardiovascular diseases. Low serum magnesium (Mg) is a predictor for cardiovascular and all-cause mortality and treating Mg deficiency may help prevent cardiovascular disease. In this review, we discuss the possible mechanisms by which Mg deficiency plays detrimental roles in cardiovascular diseases and review the results of clinical trials of Mg supplementation for heart failure, arrhythmias and other cardiovascular diseases. Cardiovascular disease (CVD) refers to a number of conditions such as heart failure (HF), arrhythmia, atherosclerosis, and stroke. Two types of HF can occur when the heart fails to pump enough blood to support other organs in the body. One is systolic HF as a result of poor contractile function, also known as HF with reduced ejection fraction (HFrEF). The other is diastolic HF as a result of reduced heart filling in diastole, also known as HF with preserved ejection fraction (HFpEF). Recently, our group has reported that magnesium (Mg) supplementation demonstrates significant protective effects on cardiac diastolic function, providing a new possible therapy for HFpEF . TherefoCVDs are the number one cause of death globally. About 650,000 Americans die from CVD each year. In the United States, about 6.5 million adults have HF, compared with ~5.7 million between 2009–2012 , in spitHypomagnesemia is commonly observed in HF ,7,8,9,10Increased consumption of processed food, filtered/deionized drinking water, and crops grown in Mg-deficient soil has led to a significant decline in Mg intake ,31,32. TMg supplementation has shown significant therapeutic effects in HF and CVD  concentration ([Mg]i) is tightly maintained in the range of 0.8–1.0 mM i could trigger Mg stores such as mitochondria i) i) ,108. Rech [Mg]i)  and Mg sfunction . Both mifunction . Therefom) i but activated under Mg deficiency i, elevated expression of cytokines , increased cardiac collagen and fibronectin content, and increased infiltration of macrophages [Mg transporters TRPM7 and MagT1 have been shown to affect and be affected by inflammation. TRPM7 is highly expressed in leukocytes and many organs such as the heart and kidney. It is a rare channel-enzyme with a channel domain to transport multiple cations (including Mg and Ca) and a kinase domain to phosphorylate serine/threonine ,166. TRPficiency ,170. Trarophages . Inhibitrophages . MagT1 conducts Mg influx and plays a key role in T cell-activated immune response ,173. MagMg deficiency, insulin resistance, and oxidative stress often co-exist in patients with diabetes, obesity, and hypertension ,175. Mg i levels and insulin sensitivity [Oxidative stress induced by Mg deficiency is a major cause of disrupted insulin signaling . Animal sitivity . On the sitivity ,186,187 sitivity ,183  . RecentlIn this section, we will review the published clinical and experimental studies on how Mg supplementation improves HF, arrhythmias, metabolic syndromes, and vascular diseases .In HF patients, hypomagnesemia is frequently observed (with a reported 7%–38% range), together with other electrolyte abnormalities such as hypocalcemia ,9. Maint4) treatment reduced left ventricular failure by 25% compared with saline placebo in the 2nd Leicester Intravenous Mg Intervention (LIMIT-2) trial [4 infusion (40 mmol over 24 h) showed significantly reduced QT variability, and the change in QT variability was correlated inversely with patient serum Mg levels [2) treatment (380 mg/day for six weeks) [4 infusion  [4 supplementation in drinking water improves diabetes-associated diastolic dysfunction in six weeks [Benefits of Mg supplementation have been reported in many studies on treating HF patients with improvements in arrhythmias, diastolic and systolic function, inflammation, and myocardial infarction rate. For example, intravenous (IV) Mg sulfate (MgSO2) trial ,193. A fg levels . Oral Mgg levels . Oral Mgx weeks)  or IV Mger 12 h) ,196 on per 12 h) . In animix weeks . 4 or MgCl2) reduces the risk of supraventricular tachycardia and VA after cardiac surgery [Mg has been used to treat different types of arrhythmias for decades, such as AF ,198,199, surgery .4 decreased the heart rate and converted to sinus rhythm patients with rapid AF [Long-term mild and moderate hypomagnesemia is associated with AF . A pilotrapid AF . Higher rapid AF . IV Mg prapid AF ,205,209 rapid AF . IV Mg arapid AF .4 infusion (50 mg/min for 2 h) eliminated the arrhythmia within 20–30 min in all cases. A single bolus of IV MgSO4 (2 g) has been shown to abolish TdP within 1–5 min in nine of 12 TdP patients [TdP is most commonly caused by medications such as QT prolonging drugs . Other cpatients . In studpatients ,213.2 intake decreased the occurrence of ventricular premature complexes, couplets and non-sustained VT in HF patients [4 administration reduced the numbers and lasting time of ventricular ectopic beats, couplets, and episodes of nonsustained VT [4  than placebo  [Mg has been shown to alleviate other VAs in many studies ,47. Oralpatients . Oral Mgpatients . IV MgSOained VT ,196. A mctively) . Intraopctively) .Mg shows protective effects on arrhythmias in MI. Low serum Mg is associated with increased risk and mortality of acute MI ,214, andi has at least two effects on L type Ca currents: inhibition of channel open probability and on channel activity under protein kinase A phosphorylation [i, the NCX current is significantly increased, compared to under physiological concentration [i mainly inhibit Ca-induced Ca release from RyR2 [i at physiological concentrations mainly plays an inhibitory role on cardiac K channels by interfering with the passage of K ions and reducing the channel open probability [The detrimental effects of Mg deficiency in arrhythmias and the protective roles of Mg supplementation have been thought traditionally to arise from Mg regulation of multiple cardiac ion channels, transporters and ionic exchangers that are responsible for the cardiac action potential, including the cardiac sodium channel , L-type rylation ,227,228.ntration ,231, whirom RyR2  by comperom RyR2 ,241. Undbability . Under Mbability ,236,237,bability . Under MThe protective effects of Mg supplementation may also result from the suppression of Mg on oxidative stress and inflammation, which are common in patients with CVD such as HF ,245 and 2, 500 mg/day for 21 weeks) improves insulin resistance [2 (26 mmol/day for three months) shows significant improvement of insulin resistance with reduced HOMA-IR index, compared with placebo [2 supplementation of pre-diabetic patients with hypomagnesemia improves glycemic control with lower fasting and post-load glucose, and higher HDL cholesterol and serum Mg after four months of treatment [Mg regulation of insulin signaling speaks to its importance in metabolic diseases such as diabetes and obesity. Mg deficiency is often reported in DM ,26,266 asistance  in part sistance . A meta-sistance . For exa placebo . Oral Mgreatment . A possil-arginine-NO system, essential fatty acids and eicosanoids. With Mg deficiency, the beneficial products such NO and resolvins are suppressed, while pro-inflammatory cytokines are elevated [Mg supplementation has shown important therapeutic effects in hypertension ,16,271 aelevated . This caelevated , and indelevated ,274, leaelevated . A recenelevated . Mg can elevated . Mg inhielevated .4 became a standard treatment for pre-eclampsia and eclampsia seven decades ago [4 decreases the risk of eclampsia to half and reduces the risk of maternal death [Mg deficiency has been reported to contribute to other vascular disorders such as pre-eclampsia and eclampsia ,280,281.ades ago ,278,279 ades ago ,283. MgSal death , and incal death ,285,286.2 shortens the atrial effective refractory period in women but prolongs the interval in men [Clinical studies have shown gender differences in Mg deficiency and associated diseases. For example, lower serum Mg is associated with DM and obesity in women but not in men ,268. Lowl in men . Mg intal in men . Generall-lactate dehydrate, Mg gluconate, MgCl2, and Mg l-aspartate hydrochloride [2 and MgSO4. Mg oxide and carbonate show extremely low bioavailability, probably because of their low solubility [Mg supplementation is normally administrated via mouth, IV or intramuscular injection. chloride . Organicchloride  or equivchloride  bioavaillubility . The oralubility . 4 and MgCl2 to acutely treat patients with arrhythmias. The dose ranges from 0.1–100 mmol/h. A high dose but short treatment for only a few minutes is often given to patients as the first bolus to boost Mg levels, followed by a significantly lower dose but lasting for hours as a prophylactic treatment. For example, in the LIMIT-2 trial, IV MgSO4 infusion was administrated at 96 mmol/h for 5 min followed by 2.7 mmol/h for 24 h to treat acute MI patients [The IV route of Mg supplementation mainly uses the inorganic salts MgSOpatients . The patpatients .Mg supplementation has beneficial effects when combined with other treatments. For example, oral Mg supplementation has been used concomitantly with antiarrhythmic drugs . IV Mg a4 (2.5 mM/h for 2 h and 1.5 mM/h for 1.5 h) was used to treat patients with TdP, and patients showed no side effects [Adverse side effects such as gastrointestinal distress, diarrhea, and nausea have been reported with Mg supplementation for both oral and IV administration ,266. For effects . For oraAnother way to maintain a normal Mg level is to increase dietary Mg intake. Recommended dietary allowances of Mg for adults are 400–420 mg for men and 310–320 mg for women . In a st4 and MgCl2) improved mortality in acute MI patients [2 on either the in-hospital or the follow-up mortality after acute myocardial infarction [Not all reports of Mg use are positive and not all preparations of Mg have the same effect. Despite efficacy on some arrhythmias, Mg showed no statistically significant effect on monomorphic VT, shock-resistant VF, and postoperative AF incidence ,317,318.patients ,219,221,farction . Moreovefarction . These cfarction ,323 and farction ,320, wheMg deficiency is common in CVD, and Mg supplementation has shown antioxidant and anti-inflammatory properties in patients with and without Mg deficiency. Mg supplementation is well tolerated with few side effects. Therefore, it may represent a reasonable additional therapy for many CVDs.


AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC–MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family—palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF;  N-arachidonoyl-ethanolamine  and 2-arachidonoyl-glycerol (2AG), the enzymatic machinery responsible for ligand synthesis and degradation, and the cannabinoid receptors 1 (CB1) and 2 (CB2) [The endocannabinoid (EC) system is composed by the lipid endogenous compounds— 2 (CB2) . CB1 and 2 (CB2) ,5. Both  2 (CB2) .As a member of the monoacyl-glycerol family, 2AG is an intermediate of diacyl- and triacyl-glycerol metabolic pathways. Due to the poor chemical stability, this compound rapidly and spontaneously isomerizes into 1AG, so that the sum of 2AG and 1AG levels (2+1AG) is often used for evaluating the 2AG biomarker potential in plasma ,8. In adN-acyl-ethanolamide (NAE) family, also including oleoyl-ethanolamine (OEA) and palmitoyl-ethanolamine (PEA) [AEA belongs to the ne (PEA) . These sne (PEA) ,11. Morene (PEA) ,12, imbane (PEA) ,13. In ane (PEA) . In partne (PEA) ,16,17. Fne (PEA)  and to sne (PEA) . PEA alsne (PEA) .Interestingly, ECs have been recently found to modulate hematopoiesis, including megakaryocyte maturation, thrombopoiesis, and platelet aggregation, as well as chemokine release and migration of immunocompetent cells ,20,21,22EC system implication in various hematological malignancies has also been investigated . In thisJAK2), myeloproliferative leukemia (MPL), or calreticulin (CALR) genes [JAK1/2 inhibitor, ruxolitinib, was introduced into clinical practice; however, many patients did not respond or did not tolerate this drug [At variance with the mentioned hematological malignancies, to date, no studies investigated the EC system role in myeloproliferative neoplasms (MPN). The MPN include clonal disorders of hemopoietic stem cells such as polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MF) that are driven by mutations in Janus kinase 2 (R) genes ; howeverhis drug . TherefoTo our best knowledge, the circulating levels of ECs and related compounds in hematological malignancies have never been reported. Here, for the first time, we investigated the circulating profile defined by levels of the sum of 2AG and 1AG and by the levels and the relative abundances of the NAE AEA, PEA, and OEA in patients affected by MPN, including ET, PV, and MF. In addition, we associated the EC and NAE profile with clinical parameters, mutational status, and disease severity to gain further insight into MPN etiology and to highlight potential disease-related biomarkers.n = 22). MPN patients were subdivided into ET (n = 5), PV (n = 9), and MF (n = 41). p = 0.129) were observed, whereas differences were detected in age among classes (p < 0.001). MF patients were older  compared to HC , ET  and PV . MPN patients were further stratified into two risk categories: 11 (20%) low-risk (age of <60 years and having no thrombosis history) and 39 (70%) high-risk (age of >60 years and having thrombosis history) patients.The cohort included 55 patients affected by MPN, recruited at the University Hospital of Bologna, and 10 healthy control (HC) volunteers from the general population. Patients were enrolled at diagnosis or after at least three months from stopping cytotoxic therapy . In particular, MF patients exhibited higher OEA/AEA and OEA/PEA ratios as compared to HC . Furthermore, we found that 2+1AG was significantly higher in MF compared to in both ET (p = 0.001) and PV patients (p = 0.030)  g. When Pp = 0.016; Circulating levels of 2+1AG and NAEs were not associated with hematological parameters such as white blood cell count, red blood cell count, hemoglobin, platelet count, and hematocrit within MPN classes; however, we observed a direct association of AEA levels with the platelet count when the overall cohort of MPN patients was considered  allowed us to perform further investigations. Notably, sex differences were observed in MF, with higher NAE and 2+1AG levels in females (n = 19) compared with in males (n = 21) , CALR (n = 14), and MPL (n = 7) mutations and by the absence of these mutations . We found that JAK2V617F-mutated MF patients had lower 2+1AG levels compared with patients carrying CALR (p = 0.006) and MPL mutations (p < 0.001)  . Notably, 2+1AG levels were also directly associated with splenomegaly in the overall MF class  b, as welThe EC system is involved in many pathophysiological processes, and its role in cancerogenesis has been postulated . The canMPN are known to be characterized by an increased pro-inflammatory status ,31,35. APrevious studies demonstrated that circulating concentrations of 2AG are increased in pro-inflammatory states ,38,39 anJAK2V617F-mutated patients displayed lower mean levels of 2+1AG compared to CALR and MPL mutation carriers. As 2AG is rapidly released in response to pro-inflammatory stimulation of immune cells [JAK2V617F VAF. Most importantly, increasing levels of this EC were associated, in the overall MF cohort, with splenomegaly, a marker of disease severity. These results led us to hypothesize that 2AG levels might be differentially regulated by the three driver-mutated genes in MF and that increasing levels are closely related to disease severity.Another relevant finding of our study relies on the association of the EC system with the mutational spectrum of MF. For instance, ne cells , our resne cells . On the Despite the fact that the role of gender in the symptomatology of MPN is still undefined ,48, BarrAltogether, our work highlights the potential involvement of the EC system in the pathophysiology of MPN, further revealing specific associations with features of MPN subclasses. Circulating levels of ECs and related compounds are part of the complex immune-neuro-endocrine system ,17,34,50In conclusion, our work involving severe and rare hematological malignancies, overall referred to as MPN, revealed for the first time disease-specific alterations of EC and NAE plasma profile, which could help in elucidating the impact of the EC and NAE systems in the pathogenesis, progression, and identification of novel therapeutic strategies.All patients and HC gave written informed consent under the approval of the local medical ethical committee of the University Hospital of Bologna (Code 7/2019/Sper/AOUBO of 01/23/2019-Comitato Etico di Area Vasta Emilia Centro), and the study was conducted in accordance with the Declaration of Helsinki. Ten HC from the general population and 55 patients affected by MPN were recruited at the University Hospital of Bologna.® tubes, Becton Dickinson, Franklin Lakes, NJ, USA) and processed within 1 h from withdrawal. Tubes were centrifuged for 15 min at 3000× g to obtain platelet-poor plasma, and the derivative was aliquoted and stored at −80 °C.Patients and HC gave blood between 8 and 10 a.m,. after overnight fasting. Blood was collected into K2 EDTA-containing tubes  505 on the 7900 HT Fast Real-Time PCR System . CALR exon 9 sequencing was performed by the next-generation sequencing (NGS) approach with GS Junior ; analysis was performed with AVA Software (GRCh38 as referenced). Rare CALR mutations identified by NGS were confirmed by Sanger sequencing. MPL mutations were investigated by the ipsogen MPLW515K/L MutaScreen Kit (Qiagen) and by Sanger sequencing (for MPLS505N and other secondary exon 10 mutations).AEA, PEA, OEA, 1AG, and 2AG plasma levels were measured by a validated in-house assay . Briefly10(x + k), whereas those showing a negative skewness were transformed according to the equation described as the squared root of (x + k). k values resulting in zero skewness after transformations were chosen. Differences in age, sex, and study-specific variables among classes were tested by T-test, ANOVA, and ANCOVA. Specifically, for MF disease, factors other than gender as platelet count (<100 × 109/L), anemia (hemoglobin: <10), peripheral blasts (≥1%), marrow fibrosis grade, large splenomegaly , and MPN-10 total symptoms score (TSS) (≥20) were considered in univariate analysis. Regression analysis was performed according to the Pearson’s correlation test. The outliers were detected using the Grubbs’s test and excluded from the analyses. p-values <0.050 were considered significant. Statistical analyses were performed by Graphpad  and by Medcalc version 18.2.1 .Mean, SD, frequency, median and range were used as descriptive statistics. PEA/AEA, OEA/AEA, and OEA/PEA molar ratios and the sum of 2AG and 1AG were computed. The normality of variable distribution was analyzed by the Kolmogorov–Smirnov test. All significantly skewed variables showing a positive skewness were transformed according to the equation written as log


The propose of this review was to summarize the advances in multi-scale surface technology of titanium implants to accelerate the osseointegration process. The several multi-scaled methods used for improving wettability, roughness, and bioactivity of implant surfaces are reviewed. In addition, macro-scale methods  and laser surface texturing (LST)), micro-scale ) and nano-scale methods  are also discussed, and these surfaces are known to have favorable properties in clinical applications. Functionalized coatings with organic and non-organic loadings suggest good prospects for the future of modern biotechnology. Nevertheless, because of high cost and low clinical validation, these partial coatings have not been commercially available so far. A large number of in vitro and in vivo investigations are necessary in order to obtain in-depth exploration about the efficiency of functional implant surfaces. The prospective titanium implants should possess the optimum chemistry, bionic characteristics, and standardized modern topographies to achieve rapid osseointegration. In recent decades, the worldwide demand for dental and orthopedic implants has grown steadily, reaching approximately $45.5 billion sales in 2014 . BrånemaConsidering the non-toxic nature and biocompatibility characteristics, titanium is one of the best choices in implant applications. Titanium shows a vast number of remarkable properties, for instance, high fatigue and corrosion resistance in biological fluids . FurtherThe purpose of this article is to report the state of the art on the multi-scale technological advancements of titanium implant surfaces to accelerate osseointegration. This review mainly focuses on innovative physicochemical procedures in multi-scale-based techniques. The physical and chemical characteristics such as wettability, roughness, and bioactivity of titanium implants in relation to biological performance is fully discussed. In this regard, the multi-scale functional coatings have the potential to increase the protein adsorption and speed up the osteogenic cell migration, angiogenesis and the early bone formation and its mineralization. Nevertheless, the optimum process parameters for various technologies still need to be clarified and will be discussed in detail in this article.3 [In general, CP Ti and its alloys are used to fabricate dental implant fixtures . Meanwhi3 . CP Ti a3 ,11. Most3 ,56,57. F3 ,58,59. C3 . The opt3  revealedThe human skeleton exhibits a hierarchical structure of the macro-, micro-, and nano-scale levels, as seen in Numerous studies ,65,66 in3DP is a well-established and versatile additive material technology that attracts the researchers’ attention due to its individuation in the fabrication of complex constructs . NowadayOver the past few decades, varieties of 3DP techniques have been thoroughly studied ,89,90,91The implant surface roughness fabricated using SLM technology : 5–20 µm)) is smoother than the EBM (Ra: 20–50 µm) counterpart, because of its smaller laser spot size and thinner layer thickness (30–50 vs. 50–70 µm), smaller powder size (average diameter 30–50 vs. 60–80 µm), and lower energy input ,95. ManyThe cytotoxicity of Ti-6Al-4V made using SLM and EBM approaches was evaluated with fibroblasts, and it was seen that there was not any significant difference between values in comparison to the negative control group . The almThere is a crucial evaluation criterion, BIC percentage; a higher BIC percentage means a greater bone ingrowth level. Experimental studies ,88,100 rStructural characteristics of scaffold have vital effects on the mechanical properties and biological performance. Several studies ,102,103 In summary, 3DP technology is used to produce biomedical metal implants with complex shapes, which promise good prospects for their clinical application in the future. The standard regulatory guidelines for additive manufactured medical devices are a prerequisite to further medical implantation. Meanwhile, the reliability and repeatability of stable physicochemical properties, biological characteristics, security, and its specifications are necessary. Especially, surface modification in the 3DP implants is an indispensable concept in order to attain high osseointegration performances. Insufficient bone formation, vascularization, contiguous infection, and implant durability are still the main challenges. Lastly, the excessive production cost of the 3DP technique limits its further development and large-scale application.Nowadays, the bonding strength between bone and titanium implants can be increased using an LST technique ,77,113. 7 µm3 VS. 0.1 × 107 µm3). Patel et al. [S. aureus) bacteria and biofilm formation hence decreases the risk of implant-associated infections [To enhance the wear resistance of Ti-6Al-4V surfaces, Kümmel et al.  producedl et al.  fabricatl et al. ,124,125.l et al. ,118 provl et al. ,120. Furl et al.  displayel et al.  showed tl et al. ,124,125.l et al.  demonstrl et al. . The saml et al. , and anol et al.  indicatefections . Biofilmfections ,131,132.S. aureus adhesion) and ultimately improving antimicrobial and rapid bone integration.The advantage of LST technology mainly involves the capability of hierarchically controlling the surface texture , array pitch, depth, and other parameters to further change the surface roughness and improve the material’s abrasion resistance, contact angle, biological properties , silicon oxide (SiO2), titanium oxide (TiO2), and calcium phosphate composition [2O3 is often applied as the blasting material and it can be dissolved in acid. Blasting material particles are very hard to remove; even after ultrasonic cleaning, acid-etching, and sterilization they can be found on the sample surface. Evidently, these abrasive particles release in the peri-implant region and interfere with the osseointegration procedure. Furthermore, there is a possibility that these particles have a role in the reduction of corrosion resistance of implant surfaces in the body fluid environment [After the processing of titanium samples to their final shape, usually, further surface treatment is required in order to roughen the surface, such as grit-blasting ,11. Fromposition ,134. Al2ironment . 2 particles with an average size of 25 μm induce a medium roughness of about 1–2 μm. A study [2 particles remarkably is higher than the machined blasting surfaces. Meanwhile, current studies [2 grit-blasting. After ten years of clinical implantation, studies [2 grit-blasted titanium implant surfaces. Clinical studies [2 grit-blasting than machined implants. A study [2 or Al2O3 particles, the BIC does not show any significant differences. However, a TiO2 grit-blasted implant has increased mechanical fixation in comparison to smooth titanium surfaces. In addition, the torque force increases with the increment of surface roughness [TiO A study  revealed studies ,137 have studies ,138 have studies ,140 have A study  has reveoughness .Calcium phosphate with its excellent compatibility, bioactivity, and biodegradability is also used as blasting media in titanium implant surfaces. Based on the crystalline type, calcium phosphates can be divided into α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). β-TCP and HA are identified as effective blasting materials, as these materials produce a clean and uniform texture on the titanium implant surface. The BIC value of calcium phosphate-treated surfaces is higher than that of machined surfaces ,144. ExpA clinical study  reported3), sulfuric acid (H2SO4), hydrofluoric acid (HF), and other combined acid solutions. In some cases, the purpose of acid-etching processing is to remove the blasting residual particles remaining from the previous grit-blasting processes on the implant surfaces. Acid-etching usually fabricates the structure of the micro-pits with pit sizes in the range 0.5–2 μm [Acid-etching is defined as a procedure of roughening titanium implant surfaces with strong acid solutions including hydrochloric acid (HCl), nitric acid  can accelerate bone ingrowth, keeping its long-term success rate [4 species. This chemical treatment roughens the titanium surface and introduces TiF4 on the surface, which in turn promotes rapid bone ingrowth [Surfaces treated using dual acid-etching  was a quite safe coating since an antibacterial characteristic was observed against E. coli and S. aureus in Ag2O coatings. Another investigation [2 and HA-TiO2 coating modified by plasma-spraying showed better hydrophilicity than the heat-treated condition, and the water contact angle was 25° and 35°, respectively. It is worth mentioning that both ZrO2 and heat treatment can enhance the hardness of material surfaces [2 (0.5 wt%)/Ag2O(2.0 wt%)-HA composite coating was 32%, while it was about 11% in the plasma-spraying HA coating group [2, Ag2O, ZrO2, ZnO, and SiO2 in a suitable content is beneficial for the antibacterial property, hardness, osteo-conduction, and early bone formation [A large number of studies ,195,196 e et al.  producedng group . Utilizaormation .In fact, the corresponding standards about the plasma-spraying treatment on titanium implants in clinical applications are briefly reported in this review paper. However, this technology still has enormous potential to develop in the future.2SO4, HF, H3PO4, HNO3) for various time spans [3+ in the membrane layer or an increase in the integrity of the membrane layer, which can lead to a significant increase in resistance, resulting in a reduced current [2 nanotubes array are shown in 2 nanotubes displayed superior corrosion resistance ability. In addition, bone-like structured TiO2 nanotubes enriched with calcium and phosphorous have enhanced osteoblastic cell functions with MG-63 cells, as is shown in Anodization technology is a mature technology to change the roughness and topographic features on the surface of titanium with many influencing variables, for instance, oxidation duration, oxidation voltage, electrolyte solution type, electrolyte solution concentration, and the subsequent heat treatment process. Nanopores and nanotubes can be induced by constant potential anodization in different acid solutions  have proven clinical efficacy. Reaching to a favorable surface morphology after surface modification plays a vital role in enhancing early osseointegration. In particular, multi-scale combined topography  can shorten the phase of bone ingrowth. Blood platelet activation, protein adsorption, three-dimensional fibrin clot cross-linking, osteogenic cell migration, collagen deposition, and bone matrix formation are the main factors affecting the osseointegration process. Wettability, roughness, and chemical composition are the bridges connecting the physicochemical properties and biological properties of titanium alloy surfaces. It’s worth noting that there are still no strict requirements and qualified standards for implant surface morphology design. In addition, a large amount of pre-clinical and clinical experiments need to be done to further ensure the security and reliability of implants using new technologies. In addition, the high cost is another limitation that creates a lot of difficulties in the clinical validation stage of implant design. The future modern implants should satisfy the following characteristics: biomimetic and standardized properties, slow rate of material release into the body environment, and low cost. Multi-scale surface treated implants show considerable potential in order to design modern implant materials with enhanced properties.


After LPS treatment, the expression of AvBD1, 2, 3, 4, 5, 9, 12, CATH1, and CATHB1 was significantly upregulated in BMCs, but no genes changed expression in CEFs. After poly(I:C) treatment, AvBD2, 11, 12, 13, CATHB1 and LEAP2 increased in both cell types; CATH2 only increased in BMCs; and AvBD3, 6, 9, 14, CATH1, CATH3, and GNLY only increased in CEFs. In addition, AvBD7, AvBD14, CATH1, CATH2, GNLY, and LEAP2 showed line-specific expression dependent upon cell type (BMC and CEF) and stimulant (LPS and poly(I:C)). The characterization of mRNA expression patterns of chicken HDPs in the present study suggests that their functions may be associated with multiple types of disease resistance in chickens.Host defense peptides (HDPs) are multifunctional immune molecules that respond to bacterial and viral pathogens. In the present study, bone marrow-derived cells (BMCs) and chicken embryonic fibroblasts (CEFs) were cultured from a Leghorn line (Ghs6) and Fayoumi line (M15.2), which are inbred chicken lines relatively susceptible and resistant to various diseases, respectively. The cells were treated by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly(I:C)) and, subsequently, mRNA expression of 20 chicken HDPs was analyzed before and after the stimulation. At homeostasis, many genes differed between the chicken lines, with the Fayoumi line having significantly higher expression ( Thousands of host defense peptides (HDPs), also known as antimicrobial peptides, have been identified in various organisms including bacteria, fungi, plants and animals ,2. HDPs LEAP2), and NK-lysin  + Line*Treatment + Line*Time[Treatment] + Replicate&Random. In the model, line included Fayoumi and Leghorn. Lipofection included the non-poly(I:C) but lipofectamine-treated and no-treatment control groups. Time included 0, 1, 3, 6, 12, and 24 h after the experiment initiation. Replicate included the effect of the IFCs and the 3 experimental replicates as a random effect.To investigate homeostatic differences between the chicken lines within each cell type, the non-poly(I:C)-treated and non-treated controls from IFC3, 4 and 5 were subset by cell type (BMC and CEF); these subsets were separately fit with the linear regression model of dCt ~ Line + Lipofection + Time + Replicate&Random. In the model, line included Fayoumi and Leghorn. Treatment was categorized into poly(I:C)-treated and non-poly(I:C)-treated groups (with lipofectamine only). Time included 0, 1, 3, 6, 12, and 24 h after the experiment initiation. Replicate included the effect of the IFCs and the 3 experimental replicates as a random effect.The poly(I:C)-treated BMCs and their non-poly(I:C)-treated controls  or the poly(I:C)-treated CEFs and their non-poly(I:C)-treated controls  were separately fit to the linear regression model of dCt ~ Line + Treatment + Time[Treatment] + Line*Treatment + Line*Time[Treatment] + Replicatep < 0.05) showed higher expression in Fayoumi compared to Leghorn -treated and non-treated) groups from the poly(I:C) experimental sets , homeostatic HDP expression levels were mainly affected by line, although a few HDP expression levels were affected by time and lipofectamine . All HDP Leghorn A. Among  in CEFs . Our datp < 0.05) to LPS or poly(I:C) in BMCs or CEFs   and CATHB1  expression significantly increased in BMCs of both lines while the expression of AvBD3 (p < 0.05), AvBD5 (p < 0.05), and CATH3 (p < 0.05) only increased in Leghorn BMCs after LPS treatment  treatment (p < 0.01), but there was no response in the Leghorn line  in a line-dependent manner.Regression models were also used to investigate the effect of treatment and revealed differences in which HDPs respond significantly ( or CEFs A. After  in CEFs A. After  in CEFs A. Expres in CEFs  and were < 0.05) B,C. Fromreatment B. Althouorn line C. There orn line . CollectAvBD1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and CATHB1, maintaining the difference between lines observed in the homeostatic state  treatment, but not at homeostasis. Similarly, AvBD1, 2, 3, 4, 5, 6, 7, 9, and 10 were expressed at greater levels in Fayoumi than Leghorn BMCs with LPS treatment (LPS + non-treated), maintaining line-dependent expression patterns from the homeostatic state. However, incorporating the LPS treatment (LPS + non-treated) did change some expression patterns in BMCs; AvBD14 became significantly higher expressed in Fayoumi than Leghorn BMCs, while CATH1, CATH2, CATHB1, GNLY, and LEAP2 were greater in Leghorn than Fayoumi BMCs only with LPS treatment  treatment ) manner.To determine whether the line differences observed under homeostatic conditions were affected by adding LPS- and/or poly(I:C)-stimulated groups, HDPs were compared between lines in BMCs or CEFs using the regression models for LPS- and non-treated groups (IFC 1 and 2) or poly(I:C)- and non-poly(I:C)-treated groups . Comparing Fayoumi to Leghorn in BMCs with poly(I:C) treatment (poly(I:C) + non-poly(I:C)-treated), Fayoumi BMCs had higher expression of ic state A,B. Addireatment A,B. Compic state A,C. Addireatment A,C. ThesThe inbred Fayoumi line included in this study has been shown to be relatively more resistant against various pathogens and environmental stresses than the inbred Leghorn line or a broiler line, including the Fayoumi line activating immune-related signaling pathways against viral infection and LPS treatment with/without heat stress ,59,60,61AvBD14, CATH2, and LEAP2 showed higher expression in Fayoumi than Leghorn. HDPs are well known as a critical component of immune responses that inhibit microbes such as bacteria and viruses and modulate the host immune system [From comparisons between the two lines under homeostatic conditions, all HDPs except e system ,8,62. Mae system ,64,65, tAvBDs generally maintained higher expression in the Fayoumi line than Leghorn line after LPS and poly(I:C) treatments, while the expression patterns of CATH1, CATH2, CATHB1, GNLY, and LEAP2 between the two lines varied according to the stimulant (LPS or poly(I:C)). Previous studies have reported that 14 AvBD genes are densely clustered on chicken chromosome 3 and their functional domains are highly conserved [LPS and poly(I:C) are well known as agonists of Toll-like receptors (TLRs), which recognize microbial pathogens such as bacteria and viruses, triggering host innate immune responses. LPS and poly(I:C) are respectively recognized by TLR4 on the host cell membrane and TLR3, which resides in endosome vesicles ,53. Afteonserved ,66,67,68onserved . TherefoGNLY, and LEAP2 showed variable expression patterns across lines, cell types, or stimulant treatments. Unlike human and mouse, which have a single cathelicidin on their genomes, chicken has four cathelicidins  [CATH1, 2, and 3 can be highly expressed in chicken bone marrow [CATHB1 was characteristically expressed in the bursa [LEAP2 can not only be widely expressed in the liver, intestine, gall bladder, kidney, and multiple reproductive organs but can also increase during embryo development [GNLY was shown to be characteristically overexpressed in spleen and the duodenal loop compared to AvBDs and CATHs [In addition to the AvBDs, the CATH family genes,  CATHB1) ,25. CATH CATHB1) ,23,24,25e marrow , while Che bursa ,25. Chicelopment ,27,69. Cnd CATHs . CollectIn conclusion, we analyzed the expression of the major chicken HDPs and confirmed the higher expression of most HDPs in the disease-resistant Fayoumi line compared to the disease-susceptible Leghorn line at homeostasis and in an LPS- or poly(I:C)-stimulated state. Thus, our results suggest that HDP functions may be associated with disease resistance mechanisms in chicken. Functions and regulatory mechanisms of these HDPs should be further studied to understand the relationship between specific pathogen resistance and HDPs in these inbred chicken lines and commercial populations.


WISP1) expression and explored its correlation with prognosis in pan-cancer using tumor IMmune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). We also examined correlations between WISP1 and immunocyte infiltration using TIMER. We identified genes co-expressed with WISP1 using the LinkedOmics database and analyzed associated gene ontology using Metascape. Finally, we constructed protein-protein interaction networks and examined correlations between genes co-expressed with WISP1 and immunocyte infiltration in pan-cancer. WISP1 level differed between human pan-cancer tissues and normal tissues, indicating its potential as a prognostic biomarker. WISP1 expression was correlated with tumor purity and immunocyte infiltration, especially monocyte-macrophage trafficking and M2 polarization. Genes co-expressed with WISP1 were mainly associated with extracellular matrix organization, with collagen members COL6A3, COL5A1, and COL8A1 being key genes correlated with macrophage infiltration and M2 polarization in pan-cancer. Conversely, in certain types of cancer with better prognoses, WISP1 was associated with low M2 macrophage infiltration. These results suggest that WISP1 affect clinical prognosis through associations with tumor purity, immune cell infiltration, and macrophage M2 polarization in pan-cancer, with collagen member proteins may serving as effector molecules of WISP1.Cancer is becoming the leading cause of death and a major public health problem. Although many advanced treatment strategies are currently in use, the general prognosis of cancer patients remains dismal due to the high frequency of recurrence, metastasis. The identification of effective biomarkers is important for predicting survival of cancer patients and improving treatment efficacy. In this study, we comprehensively analyzed WNT1-inducible-signaling pathway protein 1 ( According to estimates from the World Health Organization (WHO) in 2015, cancer is the first or second leading cause of death, with increasing incidence and mortality . In ChinThe CCN protein family in humans comprises six cysteine-rich regulatory proteins that are implicated as multitasking signal integrators in cancer . MembersWISP1 mRNA expression and explored its correlation with prognosis in pan-cancer using Tumor IMmune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). We examined the correlation between WISP1 expression and immunocyte infiltration using TIMER. We examined genes co-expressed with WISP1 using the LinkedOmics database and analyzed associated gene ontologies (GO) using Metascape. Finally, we constructed protein-protein interaction (PPI) networks and focused on genes co-expressed with WISP1 to explore its association with immunocyte infiltration in pan-cancer. Our findings suggest that WISP1 affects clinical prognosis through associations with tumor purity, immune cell infiltration, and macrophage M2 polarization in pan-cancer, with collagen member proteins most likely serving as WISP1 effector molecules.In the present study, we comprehensively analyzed 1  via the “Diff Exp” module in TIMER. Correlations between WISP1 expression and immune cell infiltration, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells, were explored via “Gene” modules in TIMER with Spearman correlations. Spearman correlations were also calculated to evaluate correlations between WISP1 expression and markers of monocytes , Tumor-associated macrophages , M1 macrophages , and M2 macrophages , as well as three collagen members  via “Correlation” modules in TIMER. Correlation outputs were in part graphically presented as heatmaps.TIMER is a comprehensive resource allowing the systematic analysis of immunocyte infiltrates across diverse cancer types1 . ExpressWISP1 expression and survival in various types of cancer. Correlations between WISP1 expression and markers of immune cell also evaluated using GEPIA22.GEPIA2 is an updated version of GEPIA for analyzing the RNA sequencing data of 9,736 tumor samples and 8,587 normal tissue samples from the TCGA and Genotype-Tissue Expression projects using a standard processing pipeline . We usedWISP1 expression and survival in diverse cancer types were analyzed using the Kaplan-Meier plotter3 , and recurrence-free survival (RFS) data were downloaded from the Gene Expression Omnibus, European Genome-phenome Archive, and TCGA databases. Correlations between plotter3 . Hazard WISP1 in various cancer types were analyzed using the LinkedOmics database4, a publicly available portal that includes multi-omics data from all 32 TCGA cancer types  pathways, were enriched using the Metascape online tool5. Overlaps between multiple gene lists at the gene and shared term levels were shown in a circos plot. Protein-protein interaction (PPI) enrichment analysis was performed using BioGrid, InWebIM, and OmniPath databases. The Molecular Complex Detection (MCODE) algorithm was applied to identify densely connected network components.Metascape is a free, well-maintained, user-friendly gene-list analysis tool for gene annotation and analysis  that fun6. And then, we merged the data using Perl software (mRNA_merge.pl). RNA-seq profiles were normalized using Voom standardized method   of HNC cohort were downloaded from the Cancer Genome Atlas (TCGA) websitel level) .7 . Upset plots were created in ImageGPWISP1 mRNA levels among diverse cancer types and adjacent normal tissues in TIMER revealed significantly higher WISP1 expression in breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck cancer (HNSC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), rectum adenocarcinoma (READ), and stomach adenocarcinoma (STAD). By contrast, WISP1 expression was significantly lower than that in adjacent normal tissues in kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), prostate carcinoma (PRAD), and uterine corpus endometrial carcinoma (UCEC) (The comparison of a (UCEC) .WISP1 and prognosis, 32 human cancers were investigated using GEPIA2. The relationship between WISP1 and prognosis varied across different types of cancers. In most cancers, high expression of WISP1 was associated with shorter OS , KIRP , LIHC , LUSC , OV , PAAD , SARC , STAD , and THCA . Patients in the WISP1-high group also had shorter RFS than those in the WISP1-low group for CESC , KIRP , LUAD , PAAD , SARC , and THCA , COAD , and PAAD ; with infiltrating levels of TAMs in 15 types of cancer, especially BLCA , COAD , and READ ; and with infiltrating levels of M2 macrophages in 16 types of cancer, especially BLCA , COAD , and PAAD . Correlations between WISP1 expression and M1 macrophages were weak or non-existent  organization in GO analysis  in which analysis .WISP1 in the selected seven cancers using Metascape. Accumulative hypergeometric p-values were calculated and shown as a heatmap for the top 20 clusters with enriched GO/KEGG terms. Genes co-expressed with WISP1 were significantly enriched in ECM organization in seven cancers , not correlated with infiltrating levels of B cells , and weakly correlated with CD8+ T cells , CD4+ T cells , macrophages , neutrophils , and dendritic cells  than those in the WISP1-low group (WISP1 expression without macrophage infiltration or M2 polarization is associated with better prognoses in UCEC.UCEC, which has a better prognosis, was used to investigate correlations between rophages . Furtherow group . These fSolid tumors are complex entities because they are surrounded by a heterogeneous array of ECM and various stromal cells . A seriein vitro cell invasion and motility as well as lung metastasis (In human cancers, levels of CCN protein expression are closely correlated with some biological characteristics and clinical features, including venous invasion, cellular differentiation, TNM stage, RFS, and OS . Althougtastasis . Thus, dIn vitro, WISP1 could induce epithelial-mesenchymal transition in tumor cells, leading to increased migration and invasion, however, in vivo, WISP1 could play different roles in diverse cancer types (In the present study, we showed that WISP1 expression differs between tumor tissues and adjacent normal tissues, suggesting its role as a prognostic biomarker depending on the type of cancer. er types . This raer types . Therefoer types . Thus, oCOL6A3, COL5A1, and COL8A1 being key genes in a majority of cancers. Collagens, which are major components of the ECM, are involved in the regulation of tumor cell proliferation, migration, and invasion. Collagen members, including COL6A3, COL5A1, and COL8A1, act as oncogenes in the progression of different types of cancer and are highly correlated with poor prognosis in cervical cancer patients (To further analyse the role of WISP1 in regulating the immune microenvironment, the top 100 genes co-expressed with WISP1 were examined in seven selected cancers using the LinkedOmics database, and enriched GO/KEGG terms were explored using Metascape. We showed that genes co-expressed with WISP1 may act as tumor promotors by regulating ECM organization, with the collagen members patients . HoweverHigh expression of WISP1 is associated with poor prognoses in various cancer types; WISP1 could serve as a prognostic biomarker when considering tumor purity and macrophage infiltration. Also, enhanced monocyte-macrophage trafficking and M2 polarization are correlated with expression of collagen members COL6A3, COL5A1, and COL8A1, which may orchestrate the cancer-promoting effects of WISP1. These findings shed light on the pro-carcinogenic role of WISP1 in pan-cancer, suggesting that WISP1 affects clinical prognosis through associations with tumor purity, immune infiltration, and macrophage M2 polarization, and collagen member proteins may serving as effector molecules.https://portal.gdc.cancer.gov/.Publicly available datasets were analyzed in this study. This data can be found here: XL, YB, ZX, FJ, FC, JL, QJ, and YL contributed to the study design and analyses. QJ conceived the study. XL and YB performed the majority of the bioinformatics analyses. ZX, FJ, FC, and JL participated in data analysis and visualization. QJ and YL drafted and prepared the manuscript. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Candida albicans. C. albicans is one of the most prevalent fungal species of the human microbiota that asymptomatically colonizes healthy individuals. However, C. albicans can also cause severe and life-threatening infections when host conditions permit . Like many other pathogens, this ability to cause infections depends, in part, on the ability to form biofilms. Once formed, C. albicans biofilms are often resistant to antifungal agents and the host immune response, and can act as reservoirs to maintain persistent infections as well as to seed new infections in a host. The majority of C. albicans clinical isolates are heterozygous (a/α) at the mating type-like (MTL) locus, which defines Candida mating types, and are capable of forming robust biofilms when cultured in vitro. These “conventional” biofilms, formed by MTL-heterozygous (a/α) cells, have been the primary focus of C. albicans biofilm research to date. Recent work in the field, however, has uncovered novel mechanisms through which biofilms are generated by C. albicans cells that are homozygous or hemizygous  at the MTL locus. In these studies, the addition of pheromones of the opposite mating type can induce the formation of specialized “sexual” biofilms, either through the addition of synthetic peptide pheromones to the culture, or in response to co-culturing of cells of the opposite mating types. Although sexual biofilms are generally less robust than conventional biofilms, they could serve as a protective niche to support genetic exchange between mating-competent cells, and thus may represent an adaptive mechanism to increase population diversity in dynamic environments. Although conventional and sexual biofilms appear functionally distinct, both types of biofilms are structurally similar, containing yeast, pseudohyphal, and hyphal cells surrounded by an extracellular matrix. Despite their structural similarities, conventional and sexual biofilms appear to be governed by distinct transcriptional networks and signaling pathways, suggesting that they may be adapted for, and responsive to, distinct environmental conditions. Here we review sexual biofilms and compare and contrast them to conventional biofilms of C. albicans.Biofilms, structured and densely packed communities of microbial cells attached to surfaces, are considered to be the natural growth state for a vast majority of microorganisms. The ability to form biofilms is an important virulence factor for most pathogens, including the opportunistic human fungal pathogen  Biofilms are communities of microbial cells that are attached to surfaces and encased in a protective substance called the extracellular matrix ,2,3,4,5.Candida albicans, which is the most commonly encountered human fungal pathogen in clinical settings [C. albicans causes a wide variety of infections, ranging from benign mucosal  to hematogenously disseminated (bloodstream) candidiasis [Candida infections are notably serious in immunocompromised individuals, such as AIDS patients, patients undergoing chemotherapy, transplantation patients receiving immunosuppression therapy, and patients with implanted medical devices [C. albicans has been ongoing for over 70 years, most work has historically focused on C. albicans in its planktonic growth state. Over the last 20 years, however, the biofilm growth state of C. albicans has become a major area of research focus. C. albicans can form biofilms on abiotic surfaces , as well as biotic surfaces  [C. albicans biofilm are protected from the host immune response, mechanical perturbations, and chemical stresses, allowing C. albicans to persist in the host and potentially cause recalcitrant infections [C. albicans biofilm has been discovered; although structurally similar to “conventional” biofilms, these “sexual” biofilms have many distinct phenotypic characteristics and generate a unique microenvironment that supports C. albicans mating [Biofilm formation is a key virulence factor for most pathogens, including settings ,4,5,6,7. devices ,9,10. Aly tract) ,4,5. Oncfections ,4,5. Mors mating ,12.C. albicans is also a normal commensal in the majority of healthy humans. Remarkably, C. albicans can asymptomatically colonize several diverse regions of the body, including the oral cavity, gastrointestinal tract, skin, and genitourinary tract of humans [C. albicans to undergo distinct morphological transitions in response to environmental cues; the best characterized examples include the yeast to hyphal cell transition and the transition between two distinct phenotypic cell types, termed “white” and “opaque” [Best known as the most common cause of life-threatening fungal infections in hospital settings, f humans ,14,15,16f humans ,18. This“opaque” . We begiN-acetylglucosamine, or nutrient limitations [White and opaque cell types are heritably maintained for many generations, and reversible switching between the two cell states occurs stochastically under standard laboratory growth conditions . This baitations ,29,30,31itations .MTL) locus [C. albicans MTL locus can carry two distinct configurations, a and α, which consist of genes that specify the a and α mating types, respectively [C. albicans clinical isolates (~97%) are diploid and exist in the MTL-heterozygous (a/α) configuration, however a few clinical isolates have been found to exist in the MTL-homozygous (a/a or α/α) configuration [MTL-heterozygous strains express the sex genes a1MTL and MTLα2, the protein products of which form a heterodimer that directly represses the white to opaque transition by binding to the promoter region of WOR1, the master regulator of the opaque cell type, and repressing its transcription [MTL-homozygous strains contain either a1MTL or MTLα2, but not both, and thus WOR1 expression is derepressed and switching to the opaque state occurs stochastically at a rate of approximately once every 104 cell divisions [The ability to undergo the white to opaque transition is controlled by the configuration of a discrete region on chromosome 5 known as the Mating Type-Like (L) locus ,34,35. Tguration ,34. MTL-cription ,36. MTL-ivisions ,33,34,36ivisions ,37.C. albicans clinical isolates are heterozygous at the MTL locus, and were previously presumed to be “locked” in the white cell state [MTL-heterozygous isolates can undergo white to opaque switching in vitro under elevated levels of CO2 and in the presence of N-acetylglucosamine, conditions that resemble that of the gastrointestinal tract; however, unlike MTL-homozygous opaque cells, these MTL-heterozygous opaque cells appear unable to mate [MTL-heterozygous cells, HBR1, which encodes a transcription factor that mediates the hemoglobin response, promotes expression of genes carried at the MTLα locus and thus indirectly reinforces the a1/α2-mediated repression of WOR1 and ultimately the repression of white to opaque switching [HBR1 in MTL-heterozygous cells results in a substantial reduction in MTLα1 and MTLα2 mRNA expression levels and a slight upregulation of a1MTL gene expression; the resulting reduction in a1/α2 heterodimer levels allows these cells to behave like a cells in regards to switching and mating [OFR1, which encodes a protein of unknown function, enables MTL-heterozygous white cells to switch to the opaque state and express both a- and α-specific pheromones and pheromone receptors, conferring ofr1 mutants with the unique ability to mate with opaque cells of any MTL configuration [MTL-homozygous clinical isolate strain P94015, which was observed to drift between “white-like” and “opaque-like” cell states, was found to contain a homozygous nonsense mutation in EFG1, which encodes a known repressor of the white to opaque transition [MTL-heterozygous cells can become MTL-homozygous through loss of heterozygosity on part or all of chromosome 5. This can occur through local gene conversion, homozygosis of an entire arm of the chromosome, or through spontaneous loss of one copy of chromosome 5 followed by duplication of the remaining homologous chromosome [Although the vast majority (~97%) of ll state ,34, rece to mate . In MTL-witching ,39. Deled mating ,39. In aguration . In addiansition . Taken tromosome ,43. Thesromosome ,45,46.MTL-heterozygous cells becoming MTL-homozygous, MTL-homozygous cells can also become MTL-heterozygous through the C. albicans parasexual life cycle [MTL-homozygous opaque cells can become MTL-heterozygous by mating with MTL-homozygous cells of the opposite mating type; this is termed heterothallic mating [MTL-heterozygous white cells undergo loss of heterozygosity at the MTL locus followed by switching to the opaque cell state [MTL-homozygous opaque cells secrete sex-specific pheromones that can cause opaque cells of the opposite mating type to extend mating projections towards the highest pheromone concentration gradient [C. albicans to rapidly adapt to variable environments and harsh conditions [C. albicans populations by preserving well-adapted genotypes, parasex can generate novel allelic combinations to allow for rapid evolution in changing environments [C. albicans to colonize diverse niches in the body and to its overall success as a commensal and pathogen [C. albicans population in vivo is thought to be MTL-heterozygous [MTL-homozygous white cells of opposite mating types undergo the multiple steps required for mating simultaneously, and within close enough proximity to detect mating pheromone, seems exceedingly low. Recent research, however, is beginning to uncover that homothallic mating occurs more frequent under specific in vitro environmental conditions, such as glucose starvation and oxidative stress, supporting the idea that homothallic mating may be more common than anticipated in vivo [MTL-homozygous white cells in response to mating pheromone [C. albicans biofilms, the multilayer structure of the sexual biofilm is such that its innermost layers are likely to contain lower levels of oxygen and nutrients than the layers closer to its surface, and thus sexual biofilms could be a niche that supports homothallic mating.In addition to fe cycle . During c mating ,48,49. Ic mating . Generalll state ,51,52,53gradient ,55. Thisronments ,55, whicpathogen ,47. Giverozygous , the pro in vivo . Intriguheromone ,12. LikeC. albicans biofilm consists of a basal layer of yeast cells with hyphae and pseudohyphae extending away from the substrate to which they are adhered [MTL-heterozygous and MTL-homozygous white cells form different types of biofilms in response to different stimuli [MTL-heterozygous (a/α) cells form robust biofilms in response to shear flow forces and various environmental conditions , and are termed conventional biofilms [MTL-homozygous (a or α) white cells in response to mating pheromone are less robust than conventional biofilms [Perhaps the most striking difference between the white and opaque cell types is that opaque cells can mate with other opaque cells, but form severely impaired biofilms, while white cells can form robust biofilms, but are unable to mate ,52,57,58 adhered ,60. In r stimuli ,57,58,59biofilms ,12, but a and α pheromones produced by C. albicans, encoded by a1MF and MFα1 respectively, play essential roles in the processes of heterothallic and homothallic mating [MFα1, producing a trimeric pheromone precursor peptide, whereas white α cells do not [a cells only weakly express a1MF under standard laboratory conditions [a cells highly express both a1MF and MFα1 [a1MF also encodes a precursor peptide which is predicted to be processed similarly to the a-pheromone of Saccharomyces cerevisiae [a pheromone precursor peptide is thought to occur via the Ste24 and Axl1 proteases [a-pheromone is a prenylated tetradecapeptide with the sequence AVRSVSTGNCCSTC, and requires Hst6, an ABC transporter, to leave the cell [a-pheromone, most pheromone signaling experiments in the field are carried out using a cells and the addition of chemically synthesized α-pheromone.The c mating ,61,62,63s do not ,66,67,68and MFα1 ,58. MFa1revisiae ,70. Initroteases . The devroteases . The matthe cell . Due to a1MF and MFα1 are expressed in a cells in response to pheromone, α-pheromone is typically degraded by Bar1, an aspartyl protease, via a phenomenon known as “barrier activity” [C. albicans to undergo auto-pheromone stimulation and thus prevents homothallic mating. Deletion of BAR1 in C. albicans allows for homothallic mating through an autocrine pathway where opaque a cells excrete α-pheromone, which then binds to Ste2, the α-pheromone receptor, on the same cell, leading to self-activation for mating [a cells to undergo homothallic mating despite high BAR1 expression levels [C. albicans as homothallism allows for same-sex mating to occur within cell mixtures of the same mating types and between certain strains that are incompatible for heterothallic mating [C. albicans relies on generating aneuploid progeny for genetic diversity, rather than recombination during meiosis, homothallic mating between clones in this capacity could rapidly and efficiently introduce genetic diversity into a population [Although both ctivity” . Barrierctivity” . It alsor mating . In addin levels , resultin levels ,56. Thesn levels ,56. Thisc mating . Given tc mating ,12. In apulation ,54,55.C. albicans employs a conserved Mitogen-Activated Protein Kinase (MAPK) signaling pathway to transduce pheromone signals and alter gene expression [a cells to recognize α-pheromone, and Ste3, expressed on α cells to recognize a-pheromone [α subunit (Cag1) from the Gβ subunit (Ste4), and the Gγ subunit (Ste18) of a heterotrimeric G-protein [STE4, CST5, CEK1, and CEK2 are expressed at lower levels in white cells than opaque cells [STE4, CST5, and CEK2 (CEK1 was not tested) at levels similar to opaque cells have been shown to undergo mating at frequencies approaching that of opaque cells [cek1 mutants mate at much lower frequencies than opaque cek2 mutants [pression ,74. Thisheromone ,74,75. A-protein ,74,75,76-protein ,74,75. A-protein ,74,75,77-protein ,73. The -protein . Interesue cells , and theue cells . It is a mutants . The pre mutants .GPA2, which encodes a G-protein α-subunit that functions at the beginning of the cyclic AMP-protein kinase A (cAMP-PKA) pathway [When opaque cells sense pheromone of the opposite mating type, they become activated for mating via the MAPK signaling pathway (described above). This pheromone stimulation can occur under a variety of different environmental conditions, including planktonic and biofilm conditions ,61,62,68C. albicans is mediated by the transcription factor Cph1, a homolog of the transcription factor Ste12 in S. cerevisiae that is activated by a MAPK signaling pathway and controls genes involved in mating [FGR23), cell fusion , karyogamy , MAPK signaling , and adhesion and virulence  [MCM6, MCM7, PRI2, and POL5a) are specifically repressed in opaque cells responding to pheromone, suggesting that exposure to pheromone further slows progression out of the G1 phase of the cell cycle [a cells, STE2 is upregulated, and the α-pheromone receptor Ste2 becomes localized to the tip of growing cellular extensions known as mating projections or conjugation tubes [The opaque cell pheromone response in n mating ,81,82,83 RBT1/4) ,58,62,68ll cycle . In opaqon tubes ,53,58,62on tubes ,53. Not on tubes . These fC. albicans white cells are unable to mate, a and α white cells still express pheromone receptors and are thought to respond to pheromone in a Cph1-dependent manner [STE2, HST6, FIG1, FUS1, KAR4), which may be an artefact derived from the co-option of Cph1 by white cells for biofilm formation [RBT1, HWP1/2, ECE1, PGA23/50, SAP5/6) [a cells do not experience a halt in their cell cycle upon exposure to α-pheromone [C. albicans has rewired aspects of cell–cell communication to be used for host–pathogen interactions, which may provide insight into the unique history of this opportunistic pathogen. Additional work on the regulatory controls of white and opaque cells may improve our understanding of how transcription factors drift to regulate novel functions.Although t manner ,58, albet manner ,68. It it manner ,68,86, sheromone ,52. Overheromone . Over thC. albicans are both composed of yeast-form, pseudohyphal, and hyphal cells [C. albicans biofilm life cycle typically begins when planktonic yeast-form cells adhere to a substrate in response to specific environmental stimuli [C. albicans biofilm matures, the cells within the biofilm produce extracellular matrix material, composed predominantly of proteins, polysaccharides, and DNA that surrounds all of the cells within the biofilm [C. albicans biofilms, the configuration of the MTL locus and the phenotypic state of the cells play important roles in determining the environmental stimuli that induce biofilm formation as well as certain unique physical characteristics of the biofilms formed. MTL-heterozygous white cells form thick and resilient conventional biofilms in response to specific environmental stimuli, such as shear flow rate and host factors, whereas MTL-homozygous white cells form thinner and weaker sexual biofilms in response to mating pheromone [Conventional and sexual biofilms formed by al cells ,12,60,86 stimuli ,5. These biofilm ,5. Once  biofilm ,5,87. Alheromone ,12,57,86C. albicans conventional and sexual biofilms acts as a physical barrier inhibiting many compounds, such as antimicrobial agents, from penetrating into the deeper layers of the biofilm [BCR1 [ALS3) in conventional biofilms, which are less  upregulated in sexual biofilms [Generally, microorganisms that exist in biofilms are protected from environmental stresses relative to microorganisms that exist planktonically . The ext biofilm ,4,5. Mat biofilm ,60. Cons biofilm ,59. Intelm [BCR1 ,59, whiclm [BCR1 ,58,59. Tbiofilms ,88,89. WC. albicans bother to form sexual biofilms in the first place? Given that ~97% of the C. albicans population in nature is thought to be MTL-heterozygous, the chance that two MTL-homozygous white cells of opposite mating types will exist in close enough proximity to undergo the complex steps involved to mate is seemingly unlikely [a cells produce their own pheromone when responding to α-pheromone, which promotes both homothallic and heterothallic mating [If sexual biofilms do not provide the same protective environment as conventional biofilms, why does unlikely . Even ifunlikely ,12. Consc mating . In termc mating ,91. Thusc mating . OverallC. albicans to coexist and interact with specific microbial species. For example, the hypoxic inner regions of conventional C. albicans biofilms support the growth of obligate anaerobic bacteria, such as Bacteroides fragilis and Clostridium perfringens [Cell heterogeneity resulting from the various microenvironments present throughout conventional biofilms is also likely to contribute to biofilm resilience . These mfringens ,93. Althfringens ,59, therC. albicans and other species (mostly bacteria) have only been studied to date within the context of conventional C. albicans biofilms. These interactions can be beneficial or antagonistic in nature. A large proportion of research to date has focused on the beneficial interactions between C. albicans and Staphylococcus species, such as Staphylococcus aureus; these two species are often co-isolated from biofilm infections with high mortality rates in clinical settings [C. albicans cells to surfaces is enhanced when C. albicans is co-inoculated with S. aureus [S. aureus and C. albicans, S. aureus cells can be found adhered to C. albicans hyphae and are present throughout the biofilm structure [S. aureus is, in fact, known to specifically recognize and bind to the adhesin Als3 on the cell surface of C. albicans hyphae, and consistent with this, cells of C. albicans als3 mutants have been found to interact with significantly fewer S. aureus cells than wild-type C. albicans cells [ALS3 expression is reduced in sexual biofilms compared to conventional biofilms [S. aureus and C. albicans are less likely to co-localize in the context of sexual biofilms. Other structural components of C. albicans biofilms are also known to play roles in mixed-species interactions. For example, β-glucans present in the extracellular matrix of C. albicans biofilms were found to aid methicillin-resistant S. aureus (MRSA) strains in surviving vancomycin, one of the few antibiotics effective against MRSA [Enterococcus faecalis can secrete EntV, a bacteriocin that inhibits conventional C. albicans biofilm formation [Pseudomonas aeruginosa can secrete a 12-carbon acyl homoserine lactone that hinders C. albicans filamentation and conventional biofilm formation by mimicking farnesol, a quorum sensing molecule produced by C. albicans that modulates filamentation [P. aeruginosa can also release phenazines that specifically inhibit C. albicans filamentation and conventional biofilm formation [Interspecies interactions within polymicrobial biofilms between settings . Althoug. aureus . In matutructure ,96,97. Sbiofilms ,60, and nst MRSA . In termormation . In anotentation ,101. P. ormation . OverallC. albicans cells, they activate a number of antimicrobial defenses, including phagocytosis, degranulation, the release of reactive oxygen species (ROS), and the release of web-like neutrophil extracellular traps (NETs) [C. albicans yeast and hyphal cells [C. albicans conventional biofilms, however, neutrophils are generally unable to penetrate beyond the outermost regions of the biofilm, ROS are not produced, and NETs are not released [C. albicans cells within a biofilm formed by the C. albicans pmr1 mutant, which is unable to produce matrix mannan [Conventional and sexual biofilms also differ in their interactions with the host immune response. Neutrophils and mononuclear leukocytes are important host players against fungal infections ,104. Whes (NETs) . In geneal cells , where treleased ,106,107.x mannan , althougC. albicans infection by phagocytosing invading cells and releasing cytokines [C. albicans cells in conventional biofilms are two to three times more resistant to killing by mononuclear leukocytes than cells growing planktonically [C. albicans cells growing in conventional biofilms are capable of altering the cytokine profile of attacking mononuclear cells [In terms of mononuclear leukocytes, these host cells typically respond to ytokines . C. albionically ,108. In ar cells ,108,109.C. albicans infection is typically initiated by the interaction of host pattern recognition receptors and pathogen-associated molecular patterns (PAMPs) and involves secretion of a variety of antimicrobial compounds. Interestingly, several characteristics of conventional and sexual biofilms inhibit the recognition of PAMPs. For example, hyphal cells, a major component of both conventional and sexual biofilms, are able to ‘mask’ the β-glucan in their cell walls, blocking a key PAMP recognized by many host immune cell types [SAP4, MSB2) are also upregulated in sexual biofilms [The host response to ll types ,110,111.ll types ,4,60. Albiofilms . NonetheMTL-heterozygous white cells grown planktonically versus in conventional biofilm conditions, and white a cells grown in sexual biofilm conditions with and without the presence of α-pheromone, there are 662 genes that are induced twofold or more in conventional biofilms, 486 genes that are induced twofold or more in sexual biofilms, and 128 genes similarly induced twofold or more in both systems  [Our current knowledge of the regulation of conventional and sexual biofilms is summarized in 5, ALS1) ,60. In a5, ALS1) ,60. The bcr1 mutant was severely defective in the rat catheter model, but formed a decent biofilm in the rat denture model, while the brg1 mutant formed normal biofilms in the catheter model, but was severely defective in the denture model) [GAL4 and RFX2 resulted in generally enhanced conventional biofilms relative to wildtype, indicating that they may serve as negative regulators of the network [cis-regulatory regions highlights a complex set of regulatory feedback loops within the core of the biofilm regulatory network [The core transcriptional network controlling conventional biofilm formation consists of nine transcription factors: Tec1, Ndt80, Rob1, Brg1, Bcr1, Efg1, Flo8, Gal4, and Rfx2 ,114. By e model) . These fe model) . Further network . Further network ,114,115. network . Althoug network ,117,118.EFG1 does not hinder sexual biofilm formation [efg1 mutant appears to form equally thick sexual biofilms relative to wildtype, indicating that EFG1 is not required for sexual biofilm formation [ndt80 mutant forms thicker sexual biofilms than wildtype, although this may not be due to Ndt80 acting as a negative regulator of sexual biofilm formation since deletion of NDT80 leads to the misregulation of cell separation genes, specifically SUN41 and CHT3 [CPH1 results in the complete obliteration of sexual biofilm formation, and it has been hypothesized that Cph1 is the terminal transcription factor activated by the MAPK cascade in both white and opaque cells responding to pheromone [Sexual biofilms are currently known to rely on four of the nine core transcription factors involved in the conventional biofilm network: Bcr1, Rob1, Brg1, and Tec1 . Deletioormation ; rather,ormation . Interesand CHT3 . This coand CHT3 ,60. Deleheromone . These iheromone . In opaqheromone ,119,120.heromone ,74,86. IFUS1 and KAR4) [The transcriptional network regulating conventional biofilms has been shown to have evolved fairly recently . By detend KAR4) . This reMTL-homozygous cells responding to mating pheromone. The physical characteristics of sexual biofilms differ dramatically from conventional biofilms; indeed, they appear to lack the major characteristics that contribute to the highly pathogenic nature of conventional biofilms. The molecular differences that result in such distinct phenotypes between the two systems remain to be determined. The significance of the unusual characteristics of sexual biofilms and their roles in the lifecycle of C. albicans is also not clearly understood. The low frequency of MTL-homozygous strains observed in nature and the apparent lack of opaque-specific niches outside of the laboratory led to questions about the existence of a parasexual lifecycle in C. albicans in nature. However, it is now appreciated that sexual biofilms may serve as a permeable and penetrable, yet protective, microenvironment that promotes mating in C. albicans. Although no in vivo model has been established to investigate the relevance of sexual biofilms in the host, the apparent disadvantageous properties of sexual biofilms for survival in the host may be outweighed by their ability to promote parasexual mating. Future work on the genetic regulation and molecular mechanisms of sexual biofilm formation will improve our understanding of the significance of sexual biofilms as well as the relevance of phenotypic switching and parasexual mating in the lifecycle of C. albicans in nature. Overall, the molecular and genetic regulation of conventional and sexual biofilm formation is quite different between the two systems. Conventional biofilms are modulated by the Ras1/cAMP signaling pathway, whereas sexual biofilms are modulated by a MAP kinase pathway; each activating a largely distinct set of transcription factors and likely different transcriptional networks. Understanding how these two transcriptional networks regulate their target genes to give rise to similar yet distinct phenotypes will also provide a basis for studies on the evolution of biofilm formation. Current and future research into sexual biofilms should provide a wealth of knowledge into the molecular genetics, pathogenesis, and evolutionary history of one of the most pervasive fungal pathogens of humans.Sexual biofilms represent a specialized kind of biofilm formed by