Conflicts of the capitolunate, causing midcarpal pain from friction can be isolated  or form part of a framework of more complex osteoarthritis phenomena . We group in the term “conflict” all of the causes (with intact cartilage or not) causing midcarpal pain by friction. Treatment by capitolunar arthrodesis can be effective, but inevitably stiffening. In other more specific cases , replacement of the proximal pole of the capitate with a synthetic implant or a tendon has shown variable results. In this work, we propose a management of these conflicts with a conservative arthroscopic technique, including capitolunate tendon interposition. We describe arthroscopic midcarpal tendon interposition (AMTI) for capitolunate conflicts. This technique prevents stiffness due to arthrodesis, but good experience in wrist arthroscopy is required to perform this operation. Arthroscopic midcarpal tendon interposition (AMTI) for avascular capitate necrosis. The patient is in supine position. The tourniquet is inflated to 250 mmHg. The traction is applied by a Finochietto hand or by Chinese fingers. Harvesting of the ipsilateral PL is performed using a stripper . The graft is harvested through a small incision at the distal flexion crease of the wrist joint at the base of the carpal tunnel. A classic arthroscopic midcarpal exploration and debridement are performed by using RMC and UMC portals. Then, the palmar midcarpal portal is used to perform this interposition with two capsular perforations: one radial (rVC) and the other ulnar (uVC). The graft is introduced from the uVC portal to the RMC portal using mosquito forceps. The tendon can be introduced from uVC to RMC or in the opposite way for the first step without it being important for the remainder of the surgery. The graft is then passed in extra-articular way from the RMC portal to UMC portal. Then, the tendon is passed from UMC portal, crossing the first strand, to the rVC portal. The graft is then knotted on itself, secured with a suture, and introduced in the midcarpal joint using a mosquito forceps. The management of capitolunate conflicts presently remains controversial, although several solutions are possible. Capitolunate arthrodesis (CLA) appears to be an effective method for the management of pain and strength, at the cost of loss of range of motion.The procedure is performed on an outpatient care basis under regional anesthesia using a tourniquet. The patient’s arm is secured to the arm board and finger traps are used to apply 5–7 kg (11–15.5 lbs) traction along the arm’s axis.First, harvesting of the ipsilateral PL is performed using a stripper . The graft is harvested through a small incision at the distal flexion crease of the wrist joint at the base of the carpal tunnel. The PL is then prepared with sutures on each free end (Vicryl 3.0).The scope  is introduced in the 3-4 portal and the shaver  in 6R portal. The first phase of the arthroscopic procedure consists of complete synovectomy with a shaver, reversing the shaver and scope positions. The scope is then introduced through the midcarpal ulnar (UMC) portal, and the shaver is introduced through the midcarpal radial (RMC) portal to debride the joint. The shaver and scope are reversed to debride the remainder of the joint. We confirmed the presence of avascular necrosis of the capitate in its proximal pole.We used a palmar midcarpal portal to perform this interposition, as described by Corella et al.The graft is introduced from the uVC portal to the RMC portal using mosquito forceps. The tendon can be introduced from uVC to RMC or in the opposite way for the first step without it being important for the remainder of the surgery .Fig 2GraThe other end of the graft is then passed in an extra-articular way from the RMC portal to UMC portal, volar to the extensor tendons . Lastly,The wrist is immobilized immediately postoperatively. The postoperative plan would include strict immobilization for 30 days, then partial immobilization for 2 weeks with some wrist movement. At 45 days, the splint was permanently removed. Rehabilitation is initiated at approximately the sixth week.At the radiocarpal level, tendon interpositions without bone excision allow a reduction of the conflict and improvement of functional signs.,Intracarpal arthrodesis are also difficult to perform, especially arthroscopically, and reoperation is not uncommon. A review of the literature, including 80 patients, revealed a reoperation rate of 14% and complication rate of 11%.,Regarding the replacement of the proximal pole of the capitate, results are too uncertain and variable to confirm effectiveness,One limitation of our study is the number of times this technique has been carried out, because of the limited number of cases. However, this technique offers a minimally invasive alternative using arthroscopy, and its conservative aspect allows a simplified return to traditional solutions that are stiffer, if the procedure fails. In addition, we performed our technique on rare pathologies to defer arthrodesis as far as possible. We believe that this technique could be applied to other capitolunate pathologies and, thus, offers alternatives to arthrodesis. Our technique presents the classic risks of wrist arthroscopies with the main drawback of performing a palmar approach, with the possible risks of nerve damage. In addition, if the posterior dissection is not performed correctly, it is possible that the interposing tendon will trap an extensor tendon, thus providing a tendon friction mechanism comparable to that of a finger pulley. Another criticism can be made if this technique is performed for cases of avascular necrosis of the head of the capitate, in that it does not treat the pathology but allows the surgeon more time to consider a less conservative surgery later and provides more stiffening. A surgeon must have good experience in wrist arthroscopy to perform this operation .Table 1S


Regular and irregular molecular brushes with polydimethylsiloxane backbone and poly-2-isopropyl-2-oxazoline side chains have been synthesized. Prepared samples differed strongly in the side chain grafting density, namely, in the ratio of the lengths of spacer between the grafting points and the side chains. The hydrodynamic properties and molecular conformation of the synthesized grafted copolymers and their behavior in aqueous solutions on heating were studied by the methods of molecular hydrodynamics and optics. It was found that the regularity and the grafting density do not affect the molecular shape of the studied samples of molecular brushes in the selective solvent. On the contrary, the grafting density is one of the most important factors determining the thermoresponsivity of grafted copolymers. It was shown that in analyzing self-organization and LCST values in aqueous solutions of poly-2-isopropyl-2-oxazolines with complex architecture, many factors should be considered. First is the molar fraction of the hydrophobic fragment and the intramolecular density. It was found that molar mass is not a factor that greatly affects the phase transition temperature of poly-2-isopropyl-2-oxazolines solutions at a passage from one molecular architecture to another. At present, stimuli-responsive polymers attract ever-increasing attention due to the wide possibilities of their practical application. They are used in biotechnology, bioengineering, and medicine, in particular, for targeted drug delivery ,2,3,4,5.In the case of water-soluble thermosensitive polymers, the response is due to a change in the hydrophilic-hydrophobic balance with temperature variation. This leads to self-organization at the molecular and supramolecular levels. For the vast majority of thermoresponsive polymers, the described processes are reversible, which makes it possible to control both the formation (or destruction) of supramolecular structures and the association of such polymers with low molecular weight compounds.Thermoresponsive polymers are divided into polymers with upper and lower critical solution temperatures  according to the nature of the response ,7. For mn-propyl-, n-butyl-, and n-nonyl-2-oxazoline), it was found that a wide temperature range of LCST (from 9 to 75 °C) can be realized  and lg Rh-D on lg M for linear PDMS and PEtOx lin were obtained by averaging the corresponding parameters for linear PDMS and PEtOx, which were calculated from the MKH equations for these polymers. With an increase in the grafted chain number in PDMSk-graft-PiPrOx molecules, the g* parameter noticeably decreases. This behavior is qualitatively similar to what is predicted theoretically etically ,68,69,70m PiPrOx . For them PiPrOx .graft-PiPrOx with the largest distance ∆L between neighboring grafted PiPrOx chains did not dissolve in water at temperatures from 5 to 65 °C in the concentration range from 0.001 to 0.01 g∙cm−3. The denser PDMS3-graft-PiPrOx brush exhibited thermoresponsivity in aqueous solutions, as illustrated in I*. The temperature T1 at which the decrease in I* begins can be considered as the temperature of the onset of phase separation. According to the data obtained by turbidimetry, the phase transition ends at T2 when I* becomes minimal.Cylindrical brush PDMS10-graft-PiPrOx solutions, there were two types of scattering objects with hydrodynamic radii Rf (particles responsible for the fast mode) and Rs (particles responsible for the slow mode). The existence of two or more types of particles in solutions of stimuli-sensitive molecular brushes was observed quite often [At low temperatures for aqueous solutions of the grafted copolymer, the distribution of light scattering intensity over hydrodynamic radii was bimodal , i.e., ite often ,72,73,74Rf and Rs was found  and coil (x = 2) for aggregates. Calculations show that the relative weight fraction c*s = cs/(cs + cf) of aggregates is about 65%, and, accordingly, the relative concentration of macromolecules c*f = cf/(cs + cf) ≈ 35% (cf and cs are weight concentration of particles responsible for the fast and slow modes).At room temperature, the contribution olecules . At the nd shape ,77:Ii ~ I (I value slowly increases with the heating of aqueous solutions of graft copolymers, while in the interval of phase separation, the rate of change of I sharply increases. Around the temperature T2, the light scattering intensity reaches its maximum value.As for other poly-2-alkyl-2-oxazolines, the thermoresponsivity of the studied polymers in aqueous solution is due to an increase in the average degree of dehydration of amide groups on heating and the formation of intra- and intermolecular hydrogen bonds ,13. In aI . The I vI on T is explained by the change in the set of scattering objects and their radii on heating (graft-PiPrOx solutions, the dehydration of poly-2-isopropyl-2-oxazoline leads to the realization of intramolecular hydrogen bonds and the compaction of macromolecules, as evidenced by the decrease in the radii Rf and Rs with temperature. Note that the relative change in the radii of molecules Rf and aggregates Rs was different. This discrepancy can be explained by the different densities of aggregates and macromolecules, i.e., the different fractions of the polymeric substance per unit volume of the scattering species. For steric reasons, macromolecules are less compact than aggregates.The dependence of  heating . For theT < T1 at all concentrations under study, I increased upon heating. This behavior is caused by a change in the weight concentrations of aggregates and macromolecules in solutions, namely, in the considered temperature range cs* due to a decrease in the fraction cf* of molecules. Accordingly, on heating, the contribution of aggregates to the integrated light scattering intensity increases, increasing the latter. Thus, at T < T1, two processes occur in aqueous solutions of PDMS3-graft-PiPrOx: both the aggregation of molecules and their compaction.A decrease in the size of the scattering objects should lead to a decrease in the light scattering intensity; however, as was mentioned above, at T1, macromolecules and aggregates with radius Rs are not detected by the dynamic light scattering, and a sharp increase in the light scattering intensity is due to the appearance of a new type of particles, giant aggregates, which radii are much larger than the size of slow mode aggregates (Rga value increases rapidly, reaching hundreds of nanometers at T → T2. Thus, in the interval T1 < T < T2, particles that were in solutions at low temperatures combined into new “giant” particles. Aggregation processes are caused by an increase in the dehydration degree of PiPrOx chains upon heating, that is, an increase in the hydrophobicity of PDMS3-graft-PiPrOx molecules with temperature.Above the temperature gregates . The RgaT2, the I values and the aggregate sizes Rga reached maximum values. Above T2, the light scattering intensity and the aggregate radii began to decrease (T > T2, compaction prevailed, but a quantitative interpretation of the results under these conditions is impossible since the solutions are turbid and the light scattering method is nonclassical.For most of the studied solutions near the temperature decrease  due to tT1 for solutions of the copolymer PDMS3-graft-PiPrOx. It seems important to study not only the behavior of an individual thermoresponsive polymer as a function of temperature and concentration but also to analyze the thermoresponsivity of a polymer class depending on the architecture parameters of macromolecules. Therefore, T1 on concentration for the PDMS4-graft-PiPrOx copolymers [graft-PiPrOx were obtained using the PDMS4 macroinitiator, in which functional undecyl tosylate groups are situated in every fourth PDMS unit. The relative grafting density for PDMS4-graft-PiPrOx samples z = 0.6 (L in PDMS3-graft-PiPrOx molecules. The Lsc/∆L ratios for PDMS3-graft-PiPrOx and sample PDMS4-graft-PiPrOx-2 differ insignificantly, while for PDMS4-graft-PiPrOx-1 with the shortest side chains, the Lsc/∆L values are approximately two times lower. For this sample, the mass fraction of hydrophobic fragments ω also differs greatly from ω for the other two PDMSk-graft-PiPrOx samples. Note that for the compared samples, the number of side chains fsc is large and differs insignificantly.polymers . Samples z = 0.6 ; accordiT1, increased with decreasing concentration. It is typical for thermosensitive polymers to dilute solutions. The rate of change of T1 with concentration is maximum in the region of strong dilution. In the region of high concentrations, the dependences of T1 on c flatten out, which makes it possible to reasonably reliably estimate the lower critical solution temperature.It is clearly seen that the temperatures, T1, it can be expected that for the studied PDMS3-graft-PiPrOx, the LCST is somewhat lower than 40 °C. This exceeds the LCST of aqueous solutions of the PDMS4-graft-PiPrOx sample with a similar M [Lsc/∆L (Lsc/∆L ratio contributes to an increase in the density of the PiPrOx shell, which shields the main PDMS chains from water molecules, and, accordingly, an increase in the LCST.Considering the behavior of the concentration dependences of imilar M  by more  [Lsc/∆L . As is kgraft-PiPrOx and PDMS4-graft-PiPrOx-2 differ slightly (graft-PiPrOx to PDMS4-graft-PiPrOx-2 can be explained by an increase in M, since the remaining characteristics for the compared samples, namely Lsc/∆L, ω, and the number of grafted chains fsc, are similar.The LCST for solutions of PDMS3-slightly . A slighgraft-PiPrOx graft copolymers with literature data for thermoresponsive linear and star-shaped PiPrOx. At a given concentration, the T1 values for aqueous solutions of PDMS3-graft-PiPrOx and PDMS4-graft-PiPrOx usually do not differ much from T1 and LCST for linear and star-shaped PiPrOx [graft-PiPrOx is an order of magnitude or higher than the usual MM of linear and star-shaped polymers. Thus, the M of the polymer is not the decisive factor determining the values of the phase separation temperatures and LCST. Probably, comparing the T1 and LCST of thermoresponsive polymers, more factors should be taken into account, for example, the hydrophilic–hydrophobic balance, the distribution of hydrophobic groups over the macromolecule volume, etc.It is interesting to compare the phase separation temperature and LCST values for PDMSk-d PiPrOx ,79, incld PiPrOx . Note thRegular and irregular polydimethylsiloxanes have been successfully synthesized. In a regular sample, functional undecyl tosylate groups are located in every third –SiO– unit, and in an irregular one, on average, in every tenth. Using these macroinitiators, molecular brushes with poly-2-isopropyl-2-oxazoline side chains were prepared, where the relative grafting density was 0.7 and 0.6 for the regular and irregular grafted copolymers, respectively.graft-PiPrOx samples in dilute solutions since they can be attributed to the class of loose molecular brushes. The shape of PDMSk-graft-PiPrOx molecules in selective solvent resembles a star-shaped molecule, the core of which is a collapsed PDMSk backbone, and the PiPrOx chains shield the core from the solvent.Using the methods of molecular hydrodynamics and optics, it was shown that the grafting density has almost no effect on the hydrodynamic and conformational behavior of the studied PDMSk-graft-PiPrOx with the largest distance between neighboring grafted PiPrOx chains not to dissolve in water, while the denser brush PDMS3-graft-PiPrOx exhibits thermoresponsive behavior in aqueous solutions. The reason for the insolubility of PDMS10-graft-PiPrOx is the hydrophobic interactions of the PDMSk backbone. In the case of PDMS3-graft-PiPrOx, these interactions lead to aggregations, and at low temperatures in aqueous solutions of this sample, in addition to isolated macromolecules, there are large loose aggregates. At heating below the phase separation temperature, the hydrodynamic radii of macromolecules and aggregates decrease in PDMS3-graft-PiPrOx solutions and the weight concentration of aggregates increases. Therefore, at T < T1, both aggregation and compaction take place. Above the temperature T1, aggregation becomes the dominant process.Decreasing the grafting density of the side chain causes the cylindrical brush PDMS10-graft-PiPrOx with the literature data for thermoresponsive linear PiPrOx, star-shaped PiPrOx, and grafted copolymers with PiPrOx chains allow us to conclude that the character of self-organization and the value of the LCST depend on numerous factors. For example, in the case of the studied PDMSk-graft-PiPrOx, the molar fraction of the hydrophobic fragment and the intramolecular density are important factors in addition to the relative side chain grafting density, more precisely, the ratio of the side chain length to the average distance between grafting points. On the other hand, in the case of poly-2-isopropyl-2oxazolines of complex architecture, the molar mass, determining the LCST for each individual polymer, is not a decisive factor when comparing the thermoresponsivity of PiPrOx with different architectures.Comparisons of the results obtained for the PDMS3-


Strategic orientation and innovation are vital determinants for accelerating the performance of small-to-medium-sized enterprises (SMEs). However, there is a lack of empirical evidence confirming the innovation at the product and process levels that instigated the SMEs’ performance. Moreover, the mediating effect of process and product innovation can play a significant role in strategic orientation and manufacturing SMEs’ performance. In this respect, this study aims to examine the mediating effect of product and process innovation between strategic orientation  and the performance of Malaysian manufacturing SMEs. The questionnaire survey gathered data from 360 manufacturing SMEs and was analyzed using partial least square structural equation modeling (PLS-SEM) to achieve these research objectives. The study analysis established that customer and entrepreneurial orientation significantly influence product and service innovation. However, the market orientation is significant for process innovation but insignificant for product-level innovation among SMEs. The study’s consequences exposed that process innovation has significantly mediated between the strategic  orientation and SMEs performance. It implies that market, entrepreneurial, and customer-related strategies would substantially improve SMEs’ performance by harnessing innovation at product and process levels. The core insights provided by the current work are to strengthen the strategic orientation that can promote product and process innovation, thereby harnessing the SMEs’ performance. Additionally, the study’s significance and limitations were reported at the end. Small-to-medium-sized enterprises (SMEs) substantially contribute to a country’s economic development by creating multiple jobs, efficiently using resources, increasing national income, and reducing poverty . As the In addition, innovation is a crucial element for any nation’s economic progression that creates competitiveness among industries and substantially influences the firm performance and broadly the economic growth of the nation . InnovatSmall-to-medium-sized enterprises contribute significantly to national growth and employment; SMEs’ performance is critical to national development . The emeIn addition, the study addresses the inadequacy of several empirical proposals and explorations related to strategic direction and innovation . ParticuThe growth of manufacturing SMEs has become more competitive through various international cross-border trade agreements such as ASEAN, TPP, and RCEP. To date, manufacturing SMEs worldwide face stiff competition, thus actively developing effective strategies to compete with cheaper alternative products from countries such as India and China . NeverthAlthough governments across the nation have formulated policies that consider SMEs’ ability to innovate, the very act of innovation remains a complex problem, especially for SMEs in emerging countries. In Malaysia, the Malaysian government runs 55 programs to infuse innovation and technology adoption to improve SMEs’ performance . The shaAchieving innovation remains a driving force among SMEs to maintain a competitive market position and achieve economic performance. However, innovation is very narrowly defined as adopting a new idea or program. Innovation can be segregated into a product, process, and business model or management level of innovation . HoweverThis work utilizes the resource-based view (RBV) to explain the firm’s internal resources instigating performance through innovation. Internal resources empower firms to gain a competitive advantage by developing capabilities for superior performance . Firm-leFirm-level innovation becomes the source of competitive advantage for the firm . The firProduct innovation describes the firm engaged in the release of new products and services to meet market demand in a dynamic marketplace . The chaProcess innovation involves using technology to change the production process or the innovative delivery of services . ProcessHowever, using technology and innovation in products and processes requires dynamic capabilities at the firm level to instigate the right innovation . ProductMarket orientation reflects both the goals and the company’s culture; it wants to remain focused on creating value for its customers to remain competitive . ObtainiSpecifically, H1a:Market orientation significantly influences production innovation.H1b:Market orientation significantly influences process innovation.Customer orientation highlights placing consumers at the center of the strategic point to bring superior business performance . Nasir eAccording to H2a:Customer orientation significantly influences production innovation.H2b:Customer orientation significantly influence process innovation.Entrepreneurial orientation reflects an organization’s tendency to explore newer market opportunities and strengthen its present market status . Such orPrecisely, in terms of product innovation, Moreover, entrepreneurial orientation plays a pivotal role in innovation by promoting values of receptiveness that constitute innovativeness . Being pH3a:Entrepreneurial orientation significantly influences product innovation.H3b:Entrepreneurial orientation significantly influences process innovation.Innovation comprises creativity, new processes, research and development, new services and products, and innovative technologies . ScholarH4a:Product innovation significantly influences enterprise performance.H4b:Process innovation significantly influences enterprise performance.In this study, market, customers, and entrepreneurial orientation were conceptualized as the main building blocks of innovation, while an association between innovation and business performance was also formulated. The study predicts that innovation plays a mediating role in the link between market, firm, and customer orientation and firms’ market performance among Malaysian manufacturing SMEs.HM1a:The relationship between market orientation and enterprise performance is significantly mediated by product innovation.HM1b:The relationship between customer orientation and enterprise performance is significantly mediated by product innovation.HM1c:The relationship between entrepreneurial orientation and enterprise performance is significantly mediated by product innovation.HM2a:The relationship between market orientation and enterprise performance is significantly mediated by process innovation.HM2b:The relationship between customer orientation and enterprise performance is significantly mediated by process innovation.HM2c:The relationship between entrepreneurial orientation and enterprise performance is significantly mediated by process innovation.Based on the above critical literature review, the following proposed hypothesized model is developed and tested in this study see .We used a cross-sectional approach to evaluate the influence of strategic focus on innovation and performance of manufacturing firms in Malaysia. Malaysian SMEs enlisted on the Malaysian Business Directory public website, provided by the SME Info Portal, formed our sampling frame . SMEs owA survey questionnaire was employed for this study. To capture “market orientation,” 10 questions items were adapted from To gage the effect of common method bias and provide procedural remedies, we constructed the items carefully and informed the respondents that the responses would be evaluated anonymously and that there existed no wrong or right answers during the data collection procedure . We assup < 0.05, which confirms non-normality in the dataset of this study. Moreover, the kurtosis coefficient for this model is 45.94, and the p-value is less than 0.05, which confirms the dataset’s non-normality.Following r2, Q2, and the effect size f2 describing the path effect from the exogenous construct to the endogenous construct. The current study had multivariate non-normality and an experimental design with a predictive nature. Therefore, we utilized the Smart PLS 3.2 to analyze the current study data.For data analysis, partial least squares structural equation modeling (PLS-SEM) was used with the Smart PLS software 3.2. PLS-SEM is a multivariate analysis instrument to gage the path models with composites’ latent constructs . Smart PUsable data were gathered from 360 Malaysian manufacturing SMEs, mostly established between 1988 and 2010. The mean time of operation was 19.49 years [standard deviation (SD) = 11.32 years], and the mean number of employees was 85.25 (SD = 57.66). In terms of types of enterprises, the data included basic metal firms (13.6%); chemicals, including petroleum (8.6%); electrical and electronics (11.4%); fabricated metal (6.1%); food, beverage, and tobacco (12.5%); machinery (5.8%); manufacture of furniture (5.6%); medical, precision, and optical instruments (2.2%); non-metallic mineral (1.9%); paper, printing, and publishing (5.6%); plastic (7.8%); recycling (5.0%); rubber (2.5%); textile, wearing apparel, and leather (1.7%); transport (0.8%); wood and other wood products (not excluding furniture) (3.9%); and others (5.0%). Lastly, most interviewees held a mid-level management position (53.9%), followed by top management (35.8), and owner/CEO (6.4%).The sales orientation, customer orientation, competitive orientation, emotional orientation, business orientation, networking orientation, sustainability orientation, and micro-enterprise sustainability are evaluated and reported in via a comparison between the loadings and cross-loadings for the tested constructs. Generally, loadings contribute an item to the latent variable it belongs to The current study used the loading and cross-loading, Fornell–Larcker Criterion, and Hetro-trait and Mono-trait (HTMT) ratio to evaluate the discriminant validity . Discrimr2 value shows that 53.1% of the total variation in product innovation is explained by market orientation, customer orientation, and entrepreneurial orientation. The f2 value of 0.405 indicates a strong effect of entrepreneurial orientation on product innovation. Finally, the Q2 value of 0.102, which is more than 0, indicates that the observed values are well reconstructed and that the model has medium predictive relevance  positive effects on product innovation. The outcome suggests accepting the H2a and H3a. The results show that the market orientation suggestively impacts process innovation, and customer orientation significantly influences process innovation. Next, the entrepreneurial orientation is significantly instigated by process innovation among the examined manufacturing SMEs. The result suggests accepting H1b, H2b, and H3b. The result is depicted in Next, the path values reported that the effect of market orientation on product innovation is also positive; however, the The coefficient for product innovation shows a significant positive effect on enterprise performance. The result suggests accepting the H4a. Lastly, the process innovation path coefficient significantly influences enterprise performance, and the finding confirms to accept H4b.p-value < 0.05) mediational effect of product innovation exists between entrepreneurial orientation and enterprise performance across the study sample. The results suggest accepting HM1c.As presented in Finally, the mediational analysis illustrates that the relationship between market orientation, customer orientation, and entrepreneurial orientation on enterprise performance is significantly mediated by process innovation. The findings support accepting HM2a, HM2b, and HM2c.Firms need sustainable performance to remain competitive and sustain in the highly competitive business environment; however, innovation is vital for superior performance for large or small enterprises. SMEs are considered less competitive as SMEs are unable to integrate innovation as a core strategic tool with the strategic orientation to become competitive . This stThe findings revealed that market orientation has a positive but not statistically significant effect on product innovation for the study sample. The study offers no support to accept H1a. The current finding of the study opposes the outcome posted by The following hypothesis examined the influence of customer orientation on product-level innovation, and the result depicted a significant and positive effect of customer orientation on product innovation. The finding achieved its support from the work of The succeeding premise inspected the impact of the entrepreneurial orientation on product-level innovation, and the consequence represented a significant and positive outcome of entrepreneurial orientation on product innovation. The current outcome realized its sustenance from Lastly, product and process innovation are found to positively impact the firm’s performance and support accepting H4a and H4b. Hence, it is logical that overall innovation has a positive and statistically significant effect on firm performance when combined. Our current study findings agree with The mediating analysis significantly influences product innovation between the relationship of strategic orientation  and the SMEs’ performance. The result offers substantial support to accept HM1b and HM1c. However, the insignificant mediating effects of product innovation exist between the market orientation and SMEs performance and offer no support to accept HM1a. Moreover, the mediating investigation reveals that the significant mediating influence of process innovation exists between the strategic orientation  on the SMEs’ performance. These outcomes suggest admitting HM2a, HM2b, and HM2c.This research provides empirical evidence on the impact of strategic orientation components  on deconstructed and unified default forms of innovation (product and process) and firm performance among Malaysian manufacturing SMEs. According to the findings, market orientation has a minor impact on product innovation but has a large impact on process innovation. Entrepreneurial and customer orientation, on the other hand, greatly activate process and product innovation.Theoretically, the study significantly contributes to the scarcity of empirical studies concerning the impact of strategic orientations by SMEs on product or process innovation and organizational performance. The study addresses the inadequacy of propositions and empirical explorations concerning strategic orientation and innovation at the product and process level , mainly In terms of practical implications, the study’s outcomes highlight that market, entrepreneurial, and customer-related strategies could help improve SMEs performance. The results suggest that policymakers should formulate and implement effective policies and programs primarily aimed at enhancing market orientation , customer orientation (consumer-centered approach), entrepreneurial orientation (exploring new market opportunities), and entrepreneurialism (exploring new market opportunities) to harness SMEs-level innovation in products and processes. It would further empower SMEs to sustain and achieve superior firm performance in the highly competitive marketplace. The result suggests that market orientation was weak among the Malaysian manufacturing SMEs and did not permit the SMEs to engage in product-level innovation. SMEs management needs to be more vigilant toward market dynamics and react to the market trendiness to rightly adjust the SMEs’ offerings to meet the changing market demands.Moreover, the lack of market orientation may not lead to the SMEs’ performance through product innovation. SMEs management is actively engaged in digital transformation to improve processes and gain competitiveness through effectiveness. The strategic orientation facilitates the firm’s preparedness to use technology for product and process improvements. SMEs management must focus on developing an entrepreneurial mindset as a critical driving force to achieve product and process-level innovation. The product and process-level innovations were transmitted to the SMEs’ performance.This study is associated with three relevant limitations. First, the work utilized three dimensions of the strategic orientation  influencing the product and service-level innovation. Future studies may consider including other strategic orientations aspects of competition, emotional, business, and networking facilitating the SMEs to engage in product and service-level innovation. Second, the work takes a general sample of SMEs to comprehensively evaluate the influence of the strategic orientation on product and service-level innovation. Future study needs to take a sample from specific industry SMEs as the SMEs’ working environment and internal working vary based on the industry. It helps offer an industry-specific understanding of the SMEs working in the industry. Moreover, the external factors also significantly influence innovation and promote local and international performance. Lastly, the current work is based on the sample taken from Malaysia. The same model may be utilized in other geographic locations to establish the holistic empirical support for the study model and apply the SMEs’ strategic orientation infusing the product innovation harnessing the SMEs’ performance sustainably.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.AA, NH, and SF engaged in research design, data collection, analysis, and reporting. AS and NZ actively played a role in analysis and wrote up. ZM played a role in drafting and management of data. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: The impact of sodium-glucose cotransporter-2 inhibitors (SGLT2is) in addition to optimal medical therapy (OMT) on the right ventricular (RV) systolic function using advanced echocardiographic analysis among outpatients with heart failure and a reduced ejection fraction (HFrEF) has thus far been poorly investigated. Methods: This was a single-center, prospective, single-blinded study in which an echocardiographic expert was blinded to the allocation of the treatment. A total of 36 outpatients with HFrEF were randomized to either OMT or OMT+SGLT2i. Both groups underwent an echocardiographic examination of the RV systolic function at the baseline and at the 3-month follow-up (3mFU). Results: The patients in both groups did not significantly differ with respect to the relevant baseline comorbidities, therapy, and clinical characteristics. The patients receiving OMT+SGLT2i showed a significant improvement from the baseline to the 3mFU in all the measured RV echocardiographic parameters, while for the OMT group, a significant improvement after the 3mFU was observed for TAPSE and s’. The mean percent change from the baseline to the 3mFU was significant when comparing OMT+SGLT2i to the OMT group concerning RV FWS , TR maxPG , and TR Vmax , respectively. Conclusions: Adding SGLT2i to OMT in patients with HFrEF resulted in a greater improvement in the RV systolic function from the baseline to the 3mFU compared to the OMT alone. Sodium-glucose co-transporter 2 inhibitors (SGLT2i) have emerged as a fundamental therapy for heart failure (HF) across the spectrum of ejection fractions. Recent results from the EMPEROR-Preserved and the DELIVER randomized trials have shown that SGLT2i  can reduce all-cause mortality and hospitalizations due to HF in patients with HF with a mildly reduced or preserved ejection fraction ,2. The gThe beneficial effects of SGLT2i on the left ventricular systolic and diastolic function, circulating natriuretic peptide levels, and functional symptom burden have been demonstrated in several studies, but not much is known about the effects of these drugs on the right ventricular (RV) systolic function ,7. The RFor these reasons, we designed the present study to investigate the potential effects of SGLT2is on the RV systolic function after it was added to optimal medical therapy (OMT) for HFrEF. This was a single-center, prospective, single-blinded study that was conducted from March 2021 to September 2021 at the Cardiovascular Disease Department, the University Hospital of Split, Croatia, in full compliance with the principles of the Declaration of Helsinki from 2013 and with the approval of the Ethics Committee of the University Hospital of Split under number 2181-147/01/06/M.S.-20-02. All the participants read and signed the informed consent form. We consecutively enrolled 36 outpatients with HFrEF according to the European Society of Cardiology guidelines (ESC) that were endorsed at the time of the initiation of the study (2016 edition)  × 100 and these values were then compared between both groups by using an independent samples t-test. The Chi-squared (χ2) test was used to examine the differences between the groups of interest with respect to the categorical variables, also for the measurements of the proportion of patients from each group that reached the cut-off value of RV FWS, which was 16% and above. Two-tailed significance values (p) were reported in all instances, while the results that reached p < 0.05 were considered to be statistically significant.All the data analyses were performed by using SPSS Statistics for Windows, version 23.0  and Prism, version 9.0.1. . Continuous data were, depending on the variable normality of distribution, shown as the mean ± standard deviation (SD) or median (interquartile range), while the categorical variables were displayed as whole numbers (N) and percentages (%). The normality of the data distribution was examined with the Kolmogorov–Smirnov test. The t-test for independent samples for variables with a normal distribution and Mann–Whitney U test for the variables with a non-normal distribution were utilized to measure the potential differences between the two groups of interest (OMT+SGLT2i vs. OMT group). The echocardiographic variables of interest were specifically examined in the “before–after” fashion in which the initial values (at the baseline) of each individual patient were pairwise compared to the values obtained at the follow-up. The mean absolute change  in these values from the baseline to the 3mFU was reported for each group of interest and these values were compared between the groups by using a t-test. Similarly, for the purpose of a main analysis, the mean percent change from the baseline to the 3mFU was calculated for each group by using formula [(Measurementn = 18) vs. OMT alone (n = 18) did not significantly differ concerning the age, sex, NYHA functional class, renal function, etiology of cardiomyopathy, relevant comorbidities , and mean daily dose or distribution of the chronic HF-related therapies, as it can be appreciated from A total of 36 consecutive HfrEF outpatients were randomized in the study. The baseline characteristics of the patients randomized to OMT+SLGT2i , s’ , 3D RVEF , RV FWS , and RV FAC . On the other hand, the improvement from the baseline to the 3mFU in the OMT-only group was significant only for the TAPSE and s’ wave , while the 3D RVEF, RV FWS, and RV FAC were all associated with a numerical improvement but failed to reach a statistical significance  and maximum pressure gradient (TR maxPG) were significantly reduced from the baseline to the 3mFU in patients that were randomized to the OMT+SGLT2i treatment . Contrary to this, no significant changes regarding the tricuspid regurgitation echocardiographic parameters were observed in the patients randomized only to OMT  from the baseline to the 3mFU demonstrated a greater numerical improvement for all the RV systolic function parameters in the OMT+SGLT2i vs. OMT group, however, a statistical significance was reached only for RV FWS, TR VTR maxPG . It is oTR maxPG A, while The TAPSE/SPAP ratio showed a mean percent increase of 48% from the baseline to the 3mFU in favor of the OMT+SGLT2i+ group compared with the OMT-group, but did not reach a statistical significance when comparing between the groups D. p = 0.084) a greater improvement from the baseline to the 3mFU in favor of the OMT+SGLT2i treatment, the s’ wave increased by 8% (p = 0.769) in the OMT+SGLT2i group, the FAC increased by the mean of 25% (p = 0.477) in the OMT+SGLT2i group, and the 3D RVEF increased by 15% more in the OMT+SGLT2i (p = 0.345) compared to the OMT-only group .Finally, as demonstrated in It should also be noted that the echocardiographic measurements at the baseline and the 3mFU visit showed a low degree of discrepancy between the expert sonographer responsible for all the measurements undertaken in the study and the control expert sonographer  of 22 mmHg) showed that PAP was reduced with empagliflozin compared with the placebo after 12 weeks of therapy, but the effects on the RV were not studied . PreviouThe exact pathophysiological mechanisms explaining these results waits to be elucidated. However, the beneficial role of SGLT2i in reducing the extent of pulmonary hypertension and RV remodeling can be explained by their multifactorial and pleiotropic effects. SGLT2i have metabolic, vascular, and hemodynamic effects. They reduce body weight due to a renal caloric loss by glycosuria, have beneficial effects on the cardiac metabolism, and improve the cardiac energetics . They alThere are some limitations to our work that should be noted. This was a single-center study enrolling an ethnically homogeneous population, thus these observations might not be able to be generalized to the overall or worldwide population. Furthermore, given that this was a “concept-generating” study that enrolled a rather small number of HFrEF patients, it is possible that some of the presented results would reach a statistical significance if a larger number of patients was enrolled. It is also possible that a timeframe greater than 3 months and a longer follow-up might be required to show a greater benefit of the addition of SGLT2i to background OMT for the improvement in the RV function in HFrEF. Similarly, a low number of patients might have generated random differences that could impact on the obtained results. Our study was not powered nor designed to capture clinical outcomes such as death and/or hospitalization events, although we can state that all patients were well-compensated at their second examination and none of them died or required hospitalization within 1 year from the follow-up examination based on our follow-up records. However, the strengths of this study are that both of the compared groups were well-matched in important baseline covariates and were treated with the same background OMT that was, correspondingly, nearly identical with respect to the daily doses. Finally, the patients were consecutively enrolled and randomly allocated a treatment, while all the measurements were recorded by the single sonographer with a high expertise in advanced echocardiography who was blinded to the allocation of the treatment. Last but not least, the echocardiographic measurements were validated against another expert sonographer.This concept-generating study demonstrated that among outpatients with HFrEF, the addition of SGLT2i to maximally uptitrated OMT resulted in a significant improvement in the RV systolic function from the baseline to the 3mFU. Moreover, a significant improvement among the OMT+SGLT2i vs. OMT-only patients was demonstrated in the parameters of the RV free wall strain and the parameters reflecting the degree of tricuspid regurgitation. Taken together, our results suggest that the addition of SGLT2i to background OMT provides an incremental benefit concerning the RV hemodynamics in outpatients with HFrEF. Further large-scale studies are welcome and warranted to confirm these initial findings.


Obtaining good-quality gluten-free products represents a technological challenge; thus, it is important to understand how and why the addition of hydrocolloids influences the properties of starch-based products. To obtain insight into the physicochemical changes imparted by hydrocolloids on gluten-free dough, we prepared several suspensions with different corn starch/potato starch/hydroxpropyl methyl cellulose/xanthan gum/water ratios. Properties of the prepared samples were determined by differential scanning calorimetry and rheometry. Samples with different corn/potato starch ratios exhibited different thermal properties. Xanthan gum and HPMC (hydroxypropyl methyl cellulose) exhibited a strong influence on the rheological properties of the mixtures since they increased the viscosity and elasticity. HPMC and xanthan gum increased the temperature of starch gelatinization, as well as they increased the viscoelasticity of the starch model system. Although the two hydrocolloids affected the properties of starch mixtures in the same direction, the magnitude of their effects was different. Our results indicate that water availability, which plays a crucial role in the starch gelatinization process, could be modified by adding hydrocolloids such as, hydroxypropyl methyl cellulose and xanthan gum. By adding comparatively small amounts of the studied hydrocolloids to starch, one can achieve similar thermo-mechanical effects by the addition of gluten. Understanding these effects of hydrocolloids could contribute to the development of better quality gluten-free bread with optimized ingredient content. Production of high quality gluten-free bread is a challenge since gluten confers unique viscoelastic properties to dough . To overThere is an increasing interest for gluten-free products in the market. People who suffer from celiac disorder avoid the gluten-containing products. Currently, gluten-free diet is the only treatment for these disorders. Gluten is important to retain gas and to obtain the desired volume, and texture of a dough system . It is eStarches are usually considered gelling materials and they significantly contribute to the texture, appearance, and overall acceptability of cereal-based foods ,33,34. DThe objective of this study was to explore the effects of three different polysaccharides ) on the thermal and rheological properties of the model gluten-free systems. Several model system formulations were prepared where ingredients were studied separately and together to better understand their role in the making of gluten-free bread.The model system formulations contained corn starch , potato starch , hydroxypropyl methylcellulose, HPMC , wheat gluten , and xanthan gum .Weight loss properties of all samples were characterized using TGA 55 . The samples were first heated from 25 to 105 °C at the rate of 20 °C/min and weight loss was followed isothermally for another 30 min. The total masses of water lost were as follows: 11.2% for corn starch, 13.1% for potato starch, 5.3% for HPMC, 7.3% for gluten, and 14.5% for xanthan gum.w/w) suspensions of corn or potato starch were prepared in water. The 40% and 60% (w/w) corn starch suspensions were prepared in 30% (v/v) ethylene glycol . Powdered starch was weighed and mixed before adding water or 30% ethylene glycol. After addition of water/30% ethylene glycol, the sample was stirred for 1 min on a vortex stirrer  at maximum speed. The sample analysis , Rheology) was performed immediately after preparation.The 20%, 30%, 40%, 50%, and 60% (w/w). Samples 1–9 were prepared by keeping the w(ingredients):w(water) = 1:1. Samples 10–12 were used to compare the viscoelastic properties of individual hydrocolloids.To prepare different model system formulations , powderepC was performed with sapphire crystal, provided by TA Instruments. Samples of model system suspension (~10 mg) were placed in hermetically sealed Tzero Aluminium pans and an identical empty pan was used as a reference.Differential Scanning Calorimetry (DSC) measurements were performed with a TA Instruments differential scanning calorimeter  and the collected data were evaluated using TA instruments TRIOS software (V5.1.1.46572). Temperature and cell constant calibration of the instrument were carried out with Indium reference samples and calibration of heat capacity All of the samples were heated from 25 to 100 °C with a heating rate of 5 °C/min. We subtracted baselines and divided the resulting heat flow by starch mass and heating rate, thus obtaining the change in heat capacity per gram of starch as a function of temperature. The observed heat effects were characterized by calculating the change in enthalpy as the area under experimental curve and transition temperature was determined as the curve peak position. Positive changes in heat capacity correspond to endothermic processes (endo up) and negative changes in heat capacity correspond to exothermic processes (exo down).G*. This modulus is comprised of the storage modulus (elastic part), G′ and loss modulus (viscous part), G″  = (29 ± 3) dL/g, we estimated the MW of xanthan gum by the Mark–Houwink relation, with α = 1.14 and K = 1.7 × 10−6 [w/v) suspensions that were stabilized in 0.1% (w/v) xanthan gum to reduce weak aggregation of granules that we observed in water-only suspensions. Approx. 5 µL of suspension was transferred to glass slide and covered by cover slip. Using the microscope Zeiss Axio Observer Z1 with 10×/0.3 objective, operating in bright field mode and equipped with an AxioCam MRm Rev.3 camera, we took at least 25 view fields of starch suspensions for each of three replicates. Using Fiji-ImageJ tools, we obtained a Feret diameter (the longest distance between any two points in the granule) of more than 2000 granules. The parameters of the obtained distribution were as follows. The mean diameter of potato starch granules was (24 ± 6) µm with a standard deviation of (17 ± 1) µm and the mean diameter of corn starch granules was (13 ± 2) µm with a standard deviation of (8 ± 1) µm.The HPMC used was labelled as K99, food grade. According to the producer specifications, K99 has M1 M NaCl ,42. A co7 × 10−6 ,42 to bep value of less than 0.05 was considered statistically significant.All samples were prepared and measured at least in triplicates. The calculated mean values and standard deviations are shown in figures unless stated otherwise. Loss factor (tan δ), which is a ratio of loss and storage modulus, was determined in linear viscoelastic range of the two moduli. Unpaired Student’s T-test, two-sided, was used to calculate the statistical significance of data sets. A w/w corn starch suspension). As the amount of available water decreased, a biphasic endothermic transition, with tailing shoulder, usually denoted asM1, was observed (w/w corn starch suspensions). As the water level was reduced, the height of the first peak diminished and consequently enthalpy of transition, ΔHtr, was reduced  = 67.0 °C, ΔHtr (20% corn starch) = 22.3 J/g), than potato starch suspensions (Ttr (20% potato starch) = 63.9 °C, ΔHtr (20% potato starch) = 49.4 J/g). This can be explained by different sizes of starch granules and amylose content. Potato starch granules are on average bigger than corn starch granules and tend to absorb higher amount of water during gelatinization [Ttr values and higher ΔHtr values [Ttr values and lower ΔHtr values [The water content and activity are, however, not the only factors affecting gelatinization process. As can be seen from nization . Consistnization ,54, the r values . Becauser values ,54,55.HC mixture (4% xanthan + 8% HPMC). Adding HPMC and xanthan gum to potato starch suspension (S2) resulted in an increase of transition temperature from 62.3 to 66.1 °C. A similar effect was observed when HC mixture was added to corn starch suspension (S4) with an increase in transition temperature from 67.4 to 70.2 °C , in transition enthalpy :m(potato starch) = 85:15) was chosen. The thermograms (Ttr(xanthan) = 2.3 °C and ΔTtr(HPMC) = 2.4 °C) when compared with the transition temperature of the starch mixture suspension without the addition of hydrocolloids. From the data in Htr(xanthan) = 0.3 J/g) or HPMC (ΔΔHtr(HPMC) = 3.5 J/g) was added to the starch mixture. Unpaired t-test showed that there was a significant difference (p < 0.05) between transition enthalpies of starch suspension and transition enthalpies of starch suspension with HPMC; thus, only ΔΔHtr(HPMC) is statistically significant. Since the mass ratio of HPMC is twice that of xanthan gum in the starch suspension (8 vs. 4% w/w), higher transition enthalpy of the starch suspension with HPMC could correspond to a higher amount of hydrocolloid. On the other hand, HPMC is less hydrophilic than xanthan gum due to its hydrophobic methoxy side groups and bulkier due to its hydroxypropyl side groups [Ttr(HC mixture) = 5.9 °C), whereas an increase in enthalpy was lower than upon addition of only HPMC and similar as upon addition of xanthan gum (ΔΔHtr(HC mixture) = 0.7 J/g). This result suggests that HPMC and xanthan gum may interact with each other and not only with starch and water.In order to investigate the individual and synergistic effects of HPMC and xanthan gum on starch gelatinization, we performed a series of experiments where HPMC and xanthan gum were added to a mixture of corn and potato starch suspension. The experimentally tested mixtures of potato and corn starch were mixtures commonly used in baking industry for gluten-free products. The ratio of potato to corn starch commonly used in the baking industry (rmograms  show thermograms a. Additie groups . BecauseHC mixture with gluten (12% w/w) (S9). HC mixture (S6). Two-state transition can be observed during heating of starch mixture with the highest thermogram peak at T = 62.5 °C (T = 64.5 °C (Htr(gluten) = 1.8 J/g). Unpaired t-test showed that there is no significant difference (p = 0.13 > 0.05) between transition enthalpies of starch suspension and transition enthalpies of the starch suspension with gluten, and thus the ΔΔHtr(gluten) value should be used cautiously. Increasing the temperature of starch gelatinization is important because higher starch gelatinization temperatures lead to higher final bread volume. If the transformation of dough from fluid aerated emulsion to a solid porous structure takes place at higher temperatures, this gives more time for the dough volume to increase, which in turn has a beneficial effect on bread quality [In order to compare the gluten-free model system with the gluten-containing system, we replaced  quality ,60,61. SIn general, the interactions between polymer molecules manifest in the rheological properties of the mixture. Amplitude sweep measurements were performed on pure hydrocolloids  and diffG′ > G″), typical for gel-like systems and entangled polymer solutions. At the same concentrations of polymers, xanthan solution had the highest tan δ ratio  also observed a G″ peak in less concentrated xanthan solutions  [G″ peaks are typically absent in linear unlinked polymers and may occur due to the relative motion between the polymer molecules, flexible end-pieces of chains and side chains, long network bridges, mobile single particles, agglomerates, or superstructures not linked or otherwise fixed in the network [Pure HPMC (S10), xanthan gum (S11), and gluten (S12) solutions have elastic character  . G″ peak network .cγ), where either G′ or G″ starts to change, marking the end of the linear viscoelastic region (LVE) region and indicating structural breakdown. The LVE was comparable for gluten and xanthan, but was much higher for HPMC. Further increase in the shear strain induced additional breaks in the gel polymer network. Eventually, at G′ = G″ (flow transition point), the samples started to behave as a liquid-like material. The difference between the shear stress at the critical point and flow point indicates how compliant the hydrocolloid structure is to shear stress. The most compliant was gluten structure, whereas xanthan and HPMC had more brittle structures. In gluten, the disintegration of the polymer network proceeded in a gradual manner and gluten retained its solid-like character at a larger span of shear stresses. If cohesive energy density (cE = ½ G′ × cγ2), a measure of structure integrity [As the shear strain increases, it reaches the critical value  at room temperature had almost no elasticity and very low viscosity , the G′ of the mixture (50 kPa) was a non-linear combination of the contribution of starch (0.01 kPa) and xanthan (0.6 kPa) elasticities, suggesting a strong interaction between the starch and xanthan hydrocolloids. The cohesive energy of this mixture was approximately 50-fold larger than in the case of starch–gluten mixture even though the concentration of the added xanthan was much lower. The addition of xanthan also removed the dilatant starch granule behavior at high shear strains.Adding xanthan to the starch granules dramatically increased stability of the mixture (S8). The increased When HPMC was added to the starch granules, the effect was even more pronounced than in the case of xanthan (S7). This mixture had approximately 80-fold higher cohesive energy density compared with the starch–xanthan mixture and 4500-fold higher than the starch–gluten mixture. Similar to the starch–xanthan mixture, the effect of starch dilatation at higher shear strains was absent. This mixture, however, was significantly less compliant to either starch–gluten or starch–xanthan mixtures. This suggests a strong interaction between starch and HPMC at room temperature.G′ was 74,000 Pa), it decreased the loss factor, and had a more compliant structure.The three-component mixture  was, in terms of cohesive energy, intermediate between the starch–xanthan and starch–HPMC mixtures. This mixture decreased the high stiffness of the starch–HPMC mixture (G′). However, the LVE critical strain, cγ, was in all mixtures significantly reduced compared with pure HPMC, xanthan gum, and gluten solutions. Adding starch to xanthan or HPMC decreased their cohesive energy density by about two orders of magnitude. In case of gluten, the cohesive energy density was further reduced for an additional order of magnitude. Additionally, the strain at the flow transition point of mixtures was significantly reduced compared with pure components. All of this indicates that rigid starch granules, when embedded into hydrocolloid network, can stabilize such network significantly. However, the stabilization effect is only evident at rest or very low shear strains; at higher strains, the starch granules weaken the structure, making it softer. In fact, this is just what is desired in quality dough. At rest, the dough should keep its shape, but for kneading it should be more compliant. Our data indicate that by fine tuning the composition of the mixture, one could in principle not only match the rheological properties of starch–gluten mixtures, but also obtain a large spectrum of rheologically different mixtures tailored for different gluten-free products.The data suggest that starch granules may interact with HPMC and xanthan hydrocolloids. The interaction is strong and may significantly harden the mixture  were studied with differential scanning calorimetry and rheology. The DSC results indicated that water availability, which plays a crucial role in the starch gelatinization process, could be modified by adding hydrocolloids. The rheology of gluten-free model systems was significantly affected by the interactions between starch and hydrocolloids. With the addition of xanthan gum and HPMC to starch, a suspension with increased viscoelasticity and increased enthalpy and temperature of starch gel transition was formed. The results indicate that by the addition of comparatively small amounts of the studied hydrocolloids to starch, one can achieve similar thermo-mechanical effects as by the addition of gluten. The addition of two hydrocolloids impacted the thermo-mechanical properties of the suspensions in the same direction, albeit with different magnitudes. This implies that the fine-tuning of dough properties by changing the amount and ratios of added HPMC and xanthan to starch mixtures is possible and could be used to optimize the production of gluten-free products.


Zirconia has been used as a reliable core material in dental restorations for years; however, its bonding to resin cement is a matter of challenge. Physical, chemical, and combinations of these techniques have been investigated to boost the properties of zirconia surface bonding. The objective of this work was to evaluate the effect of hydroxyapatite coating as a chemical therapy in combination with physical modifications on the microshear bond strength of the resin cement over zirconia. 2O3 particle abrasion (group 1), HA coating (group 2), Al2O3 particle abrasion + HA coating (group 3), and Er, Cr: YSGG laser irradiation + HA coating (group 4). The microshear bond strength was determined by bonding the blocks to the resin cement.  In the present research, 60 sintered zirconia blocks (4 × 4 × 4 mm) were randomized into four groups of 15, including Alp > 0.05).  The bond strengths (mean ± standard deviation) of modified zirconia surfaces were 16.93 ± 4.94 MPa, 16.14 ± 5.4 MPa, 19.4 ± 5.27 MPa, and 16.21 ± 3.7 MPa in groups 1–4, respectively. Test results of the ANOVA test revealed no significant difference regarding the bond strength values of zirconia surfaces to the resin cement between the studied preparation modalities ( Observations from the present study showed that HA coating can be as effective as the air-borne particle abrasion technique in improving bond strength to zirconia surfaces. Moreover, sandblasting by an aluminum oxide or Er, Cr: YSGG laser irradiation prior to HA coating of zirconia showed no significant effect on the reinforcement of bond strength values when compared to HA coating alone. The clinic hydroxyapatite coating alone or in combination with physical treatments improves the bond strength of zirconia to resin cement. Referring to the findings, the Er, Cr: YSGG lasers can generate comparable surface roughness rather than acid etching of dentin or enamel surfaces [μSBS) of zirconia to resin cement (Panavia F 2.0). There were two null hypotheses: (1) there is no difference in the μSBS among the study groups. (2) There is no difference in the patterns of failure.All-ceramic materials optimized for mechanical properties have recently attracted special attention for use in the manufacture of fixed metal-free restorations. Among these, zirconium oxide  under a pressure of 4 kg/cm2 and a distance of 10 mm for 15 s in a MESTRA sandblast apparatus , followed by cleaning with ethanol (96%) for 10 minutes in an ultrasonic cleaner. Group 2 consisted of zirconia blocks whose surfaces were coated via the HA thermal protocol so that a slurry solution was prepared by adding 10 gr of nanoparticle HA powder (less than 100 nm)  to distilled water (50 cc). Then, 1 gr of polyvinyl alcohol  was also added as the binder of the suspension, followed by heating on a magnet stirrer with 1000 rpm at 100°C for 60 min to achieve a uniform suspension. At last, the zirconia blocks were placed in this slurry at an angle of 45° for 5 s.Group 1 consisted of zirconia blocks sandblasted with AlGroup 3 consisted of zirconia blocks sandblasted with the same condition as described in group 1 and then layered with a coating of HA following the same procedure as described in group 2.μm, 140 μs pulse duration, a 20 Hz repetition rate, and 4 W output power. The laser optical fiber (with a diameter of 600 μm and a length of 6 mm) with the gold handpiece was positioned to the surface at a distance of 10 mm perpendicularly and moved in a sweeping manner manually within a 30 s exposure time over the whole area. Continuous water (55%) and air (65%) flows were applied while performing irradiation. After laser treatment, the surface was coated with HA, as explained for group 2.Group 4 consisted of zirconia blocks whose surfaces were first exposed to Er, Cr: YSGG laser irradiation with the following details: a wavelength of 2.78 S (MPa) = F (N)/A (mm). Following the shear experiment, an SEM was used to determine the fractured surfaces for achieving the failure variants that included cohesive failure, adhesive failure, and mixed failure.Subsequent to the surface treatment, all blocks in groups 2, 3, and 4 were sintered following this protocol: ambient temperature to 300°C for 10 minutes, 300–600°C for 10 minutes, 600–900°C for 30 minutes, 900–1200°C for 40 minutes, maintenance at 1200°C for 120 minutes, and cooling for annealing, employing a CWF furnace . Following the surface treatment in all study groups, the mold was selected to be Tygon Norton Performance Plastic tubes with an inner diameter of 0.8 mm and a height of 1 mm  for the Panavia F 2.0 cement bonding  to the prepared surfaces. Then, the tubes were removed with a heated, sharp scalpel. All samples were placed in distilled water for 24 hours, incubated  at 37°C, and positioned in a microtensile tester  for the determination of microshear bond strength. A metal loop  was put around the cement cylinder in the bonding site for the exerted tensile load conversion to shear load through vertical soldering of casting molds to the jig, as shown in α or p value) was considered equal to 0.05.To analyze the data, the SPSS software version 21.0 was used. The statistical comparison of the bond strength was conducted with a one-sided analysis of variance (ANOVA) among the 4 groups. In the present study, the type 1 error rate . Since there were no significant differences as determined by the ANOVA analysis, no pairwise comparison was performed between the groups.The mean ectively . AccordiThe most common failure modes in all study groups were the adhesive types. All cohesive fractures occurred in the cement cylinder. The mixed type of failure was the less common type of fracture in groups 1 and 3. μSBS was not significantly different among the study groups. While Y-TZP offers many advantages, such as high strength, fracture toughness, and good wear resistance, there are some disadvantages, such as the nonpolar nature, which leads to negligible bonding capacity with dental structure and/or overlaying ceramics [According to the mineral tooth structure, which is mainly composed of hydroxyapatite crystals, and the favorable resin cement bond strength to such a structure, it is hypothesized that coating the zirconia surface with the nanosized HA might boost the bond strength with resin cement. The current effort investigates the influence of hydroxyapatite coatings solely and in combination with physical surface modification via Er, Cr: YSGG laser irradiation, and air-borne particle abrasion. In our research line, Azari et al.  were jusceramics . Multiplceramics . The surceramics . As repoceramics .Over the years, using digital technologies in dentistry has developed. In a study by Pagano et al. , CAD/CAMμSBS values of all study groups were beyond the acceptable range, and their differences were not statistically significant. As stated by Ranjbar Omidi et al., silica coating through Cojet sand presented substantially greater μ-shear bond strength compared to Er: YAG laser and sandblasted groups. Moreover, the μSBS values of the sandblasted group were higher than those of the Er: YAG laser [2O3 air abraded + Z-PRIME Plus group. According to the findings of these two studies and other similar studies [μSBS. Although the μSBS value was higher in group 3 in comparison to group 1, such a difference was not statistically significant. This might be due to the replacement of the surface irregularities with nanosized HA, which would leave no efficient indentation for micromechanical retention of the cement layer. We coated the surface of zirconia with nano-HA on the basis of a thermal protocol. Nanoparticles provided a thin and uniform controllable coating layer with adequate strength. In addition, Abdulkader and Aljubori reported that the addition of nanohydroxyapatite (nHAp) to self-adhesive resin cement reinforced the mechanical features and bond strength of zirconia [2O3 particle abrasion. An SEM observation revealed more surface irregularities with 50 μm Al2O3 abrasion than the Er, Cr: YSGG laser (2 W and 3 W) treated Y-TZP surface [μSBS in group 3 of the present study, in comparison to group 4, could be the result of better micromechanical bonds. While several studies have investigated the impact of Al2O3 particle abrasion and its influencing factors on the sintered zirconia surface roughness [μm Al2O3 particle abrasion could result in elevated flexural strength because of the formation of compressive fields owing to the induced tetragonal–monoclinic transformation of the crystals present in the surface [There are diverse coating approaches available for exploiting the merits of HA. No standard instructions have been issued so far for the various dimensions of a particular approach, such as surface profile, porosity, texture, coating thickness, and crystallinity or crystal size effects. Several methods of coating a HA layer onto bioinert metallic implant surfaces have been previously reported, which include the plasma spray and sol-gel methods. The present work benefited from the thermal coating approach because of its fine mechanical profiles, practical feasibility, and admirable phase composition . The theAG laser . SandblaAG laser . Tanış aAG laser  indicateAG laser  reported studies , it mighzirconia , 31. The surface . Althougoughness . In the  surface . However surface . In a wo surface , the pho surface . Hence,  surface . The pre surface , 29. Sim surface . SEM obs surface .The main limitation of this research was achieving a uniform HA coating on the zirconia. It was a technique-sensitive procedure. The most common test methods used to evaluate adhesive bonding between two materials are tensile, microtensile, and shear bond strength tests . The sheμSBS of zirconia were improved by HA coating surface treatmentThe μSBS with HA surface treatment is comparable to air-borne particle abrasionImprovement of To improve the bond strength, there is no significant additional benefit from combining air-borne particle abrasion or laser irradiation with HA coatingHA coating might be a promising alternative method for air-borne particle abrasion on zirconia surfacesThe following conclusions were obtained in the present work:


Deinococcus radiodurans. In the absenceof light, we use an enhanced sampling molecular dynamics (MD) methodto overcome the photoisomerization energy barrier. We find that thecalculated free energy (FE) barriers between essential metastablestates agree with spectroscopic results. We show that the enhanceddynamics of the BV chromophore in BphP contributes to triggering nanometer-scaleconformational movements that propagate by two experimentally determinedsignal transduction pathways. Most importantly, we describe how themetastable states enable a thermal transition known as the dark reversionbetween Pfr and Pr, through a previously unknown intermediate stateof Pfr. We present the heterogeneity of temperature-dependent Pfrstates at the atomistic level. This work paves a way toward understandingthe complete mechanism of the photoisomerization of a bilin-like chromophorein phytochromes.The ability of phytochromesto act as photoswitches in plants andmicroorganisms depends on interactions between a bilin-like chromophoreand a host protein. The interconversion occurs between the spectrallydistinct red (Pr) and far-red (Pfr) conformers. This conformationalchange is triggered by the photoisomerization of the chromophore D-ringpyrrole. In this study, as a representative example of a phytochrome-bilinsystem, we consider biliverdin IXα (BV) bound to bacteriophytochrome(BphP) from  At the atomistic level, a series of conformational changesin light-responsive proteins, referred to as the protein photocycle,is triggered by absorbing the energy of a photon. One example of complexesin which photoisomerization can induce a large conformational changein the protein environment is the phytochrome family which representsa diverse class of photoreceptors present in plants, and fungal andbacterial kingdoms.4 The phytochrome family uses alineage of the GAF domain that binds a thioether-linked tetrapyrrolechromophore for light sensing.8 Phytochromes play an essential role in light-regulatedprocesses, e.g., phototaxis, pigmentation, and photosynthesis, andare interesting also as targets for optogenetic applications, imagingin tissues, light-dependent gene expression, and possible medicalapplications.14 BphP in the Deinococcus radiodurans cells, in additionto its responsibility for pigmentation and growth, is potentiallyinvolved in the resistance of these bacteria to ionizing radiationand mutagenic factors by participating in the DNA damage response.15Isomerizationis the hallmark of light-activated molecular switches.17 These forms are based on theirrespective absorption maxima in the red and far-red spectral regions.The corresponding transition is triggered by the ZZZ to ZZE (Pr to Pfr) or the ZZE to ZZZ (Pfr to Pr) photoisomerization.2 Subsequently, the event leads to a secondary structure change ofthe flexible and partially disordered tongue region and to the straighteningof the α-helix spine  but it isnot clear if a single-bond rotation is also possible (C14–C15).37 Overall, the structural and thermodynamical determinants of photoreceptionin phytochromes are still poorly understood.38The understanding of the phytochrome family has been largely drivenby experimental studies employing IR,39 caused mainly due to the existence of long time scalesinvolved in the dynamics of photoproducts.20 To achieve such a challenging goal, we employ enhanced samplingMD simulations to overcome the FE barrier of the primary photoisomerizationevent and to efficiently sample nonequilibrium configurations visitedby the BphP-BV complex in the Pr and Pfr conformers. By performingsimulations with an aggregate time of ∼2 μs, we studythe Pr and Pfr conformers starting from their equilibrium configurations,i.e., before the photoisomerization takes place (Pr) and after theprimary photochemical event is concluded (Pfr). The ultrafast photochemicalsteps during the primary photoisomerization event  cannot be observed here. However, regardless ofthe transitions between the short-lived intermediates during the photoisomerization,44 these steps lead to either Pfr or Pr which we can study using enhancedsampling MD.The goal of this article is to study, from the thermodynamicalviewpoint, the early stages of the mechanical signal transductionin the BphP-BV complex and the metastable states of Pr and Pfr photoproductsthrough MD simulations. However, this goal poses a major difficultydue to the so-called the Boltzmann sampling problemIn this paper, (i) we show that Pr and Pfr conformersare characterizedby distinctly different FE landscapes in which metastable states allowtemperature-dependent transitions related to the dark thermal reversion.This transition proceeds through a previously unknown intermediatestate of Pfr caused by the rotation of Arg-466, His-467, and Ser-468from the tongue region. (ii) We characterize FE barriers between metastablestates accompanying the Pr to Pfr transition and find they agree withspectroscopic results. (iii) We reveal the fundamental thermodynamicalrole of a protein environment acting as a selectivity filter for deepeningand widening important metastable states in Pfr. (iv) Finally, wefind that, the enhancement of the local fluctuations of the BV chromophorecontributes to nanometer-scale conformational movements in BphP, followingtwo experimentally determined signal transduction pathways. In ouropinion, this work represents an important step toward understandingthermodynamical characteristics of chromophore-bound canonical phytochromesin the Pr and Pfr states.22.1gromacs 2018.1 code45 patched with the plumed 2.8 plugin.47 As the starting structures of the BphP-BV complexes . Subsequently, unbiased MD simulationsare carried out for 200 ns for each protein–ligand complexto check the level of thermal fluctuations of the equilibrium metastablestates.We perform all MD simulationsusing the omplexes 1, we useomplexes ) and Pfromplexes ). The crfrom ref , where td in ref . We refe2.253 (VES) as implemented in theVES module available in plumed.47 The parameters used to launch the simulations remain the same asfor the unbiased simulations  = ∑kαkeikz, where α =  arereal variational coefficients and z ≡  represents the set of the biased variables as depicted in N = 10, theperiodic domain of the basis set as ,and k ≡ , where each term goes from −N to N. We employ a uniform distributionas the target probability distribution, pT, that should be obtained once the simulations areconverged. The functional Ω[Vα] (see Section S3 for a definition)is optimized using a version of stochastic gradient descent54 (SGD) with a learning rate μ = 0.1 anda stride of 1000 steps. During the analysis stage, we discard thefirst 10 ns of the simulations to ensure that we avoid the periodat the beginning of the simulations where the statistical weights, βVe, may not be equilibrated due to initialfast changes in the bias values. The remaining samples are used inthe construction of FE landscapes. The weights are scaled to the range0 to 1 to avoid numerical issues. A detailed introduction to VES canbe found in Section S3.Werun enhanced sampling MD simulations using variationally enhancedsamplingulations 2.1. The 2.3gromacs 2018.145 and plumed 2.8.47 The data supportingthe results of this study are openly available from plumed nest and Pfr (PDB: 4O01) conformers,17 sometimes referred toas the dark and illuminated forms, respectively. Both crystallographicstructures comprise the core photosensory module and the BV chromophore covalently attached to the side chain of Cys-24via a thioether linkage with the A-ring pyrrole . Upon the transition from pyrroles 1. Thus, pyrroles 1 involviC14–C15, 1b becausFigure S2). Next, we move to enhanced sampling simulations that arerun at 300 K for 500 ns for both conformers of the protein–chromophorecomplex and 200 ns simulations for the solvated chromophore. For furtherdetails regarding parameters used to run the unbiased MD and enhancedsampling simulations, see After selecting the CVs, we run 200 ns of unbiased MD simulationsat 300 K to check whether the systems are stable in the Pr and Pfrforms and observe the magnitude of the equilibrium fluctuations ofthe CVs in the X-ray conformations of Pr and Pfr. We find that during200 ns the thermal fluctuations of Pr and Pfr do not allow for thesampling of other metastable states. The systems are kinetically restrictedto the basins of the X-ray structures ], is minimized during the simulation toestimate FE, F(z). The biasing potential V(z) acts in the CV space and is representedby a selected combination of basis functions. At convergence, FE canbe estimated using umbrella reweighting.57 As such, VES is an efficient method for exploring long-time scaleevents.58 For conciseness, the theory behind CV-basedenhanced sampling and a detailed discussion of the method used forthe simulations are provided in Section S3.To drive the X-ray structures out of equilibriumand reconstruct FE landscapes, we choose an enhanced sampling MD approachknown as variationally enhanced sampling (VES).Figures S3 and S4. The remainingsamples are used to calculate the FE landscapes. In 57 We calculate also the FE profiles of the systems along the Φand Ψ dihedral angles by integrating out the unused CVs. Aswe analyze the difference between global FE minima and maxima in bothPr and Pfr, we place the deepest FE minimum at 0 kJ/mol in each landscape.As typically statistical weights in the initial stages ofenhanced sampling simulations are not equilibrated, we empty our simulationsof possible sampling artifacts performing a standard preprocessingof the gathered CV samples. Namely, we take into account only theCV samples starting from 10 ns to exclude possibly unreliable samples.We can see that after 10 ns, the weights are more stabilized as ispresented in Figure S5). For the BV chromophore in solvent,the convergence is obtained earlier . At the end of the simulations, the transitions across the CV spaceare more frequent as the bias potential renders the dynamics closeto diffusive. Additionally, to check the relative changes betweenthe FE landscapes of the protein-chromophore complexes in the Pr andPfr forms, we employ a statistical distance based on the Kullback–Leiblerdivergence to quantify the averaged difference in the whole FE landscapescalculated at different time steps of the simulations. We can seethe distance reaches a plateau at 320 ns . Additionally, the mean standard deviations of the FElandscapes calculated over the last 200 ns are 1.76 and 0.52 kJ/molfor Pr and Pfr, respectively. Taking the above into consideration,we conclude that the simulations reach the level of convergence requiredfor the further analysis.To assess the convergence of the enhanced sampling simulations,we check if the simulations efficiently explore the CV space. ForBphP-BV, after 500 ns, both simulations cross the CV space multipletimes as can be observed from the time-series of the CVs (ZZZ (ZZE (Sm (middle), Sv , Sh , and Sc (corner). The Sh and Sm metastable statesare also referred to as the Pr X-ray and Pfr X-ray states, respectively.We label the Sm metastablestate as a single superstate rather than two separate states as itconsists of two substates between which transitions are frequent enough to compare the FE between the correspondingmetastable states in both conformers. Calculating FE differences requiresa selection of CV samples from the metastable states. We perform thisby first picking only the CV samples belonging primarily to the metastablestates using a threshold value to sieve only CV samples with highstatistical weights. The remaining samples are negligible as theirweight decreases exponentially with the energy according to a Boltzmannfactor. Next, we use clustering to group the important CV samplesinto metastable states. Based on this protocol, we can easily calculatethe FE differences between the respective metastable states in Prand Pfr. We plot these differences in Figure S9b. Additional details regarding the analysis of clustering and theemployed protocol can be found in Figure S10 and Figure S11.Next, we move towardcalculating more quantitative differences between the FE landscapesof Pr and Pfr. To this aim, we use a formula for FE differences . As we calculate the differencesby comparing Pr to Pfr, therefore, a negative value means that a stateis more populated in Pr, while a positive value indicates that a stateis more populated in Pfr. The highest FE difference is between Sm states, reaching around 25kJ/mol. This metastable state has a lower FE basin in Pfr than inPr which can be explained by the fact that Sm is the state to which the Pfr X-ray structurebelongs. Slightly lower differences (15–20 kJ/mol) are shownfor Sh and Sc. This is especially interesting as Sh is the X-ray state of Pfr.The only state more favored for Pr is Sc as it is characterized by lower FE than Sc in Pfr . These findings corroborate our previous analysis consists of two substatesbetween which transitions are relatively frequent (Sm state of Pr which is notas populated as its X-ray state (Sh). We can see that one possible pathway is joining the X-raysubstate of Pfr, going through the intermediate state and the Sh metastable state which isof similar FE to that of the X-ray state of Pr. Such a transitioncorresponds to the dark thermal reversion from Pfr to Pr. It is moreprobable than the transition from the X-ray state of Pr to the Sm metastable state as the FEbarrier separating these states is much higher for Pr. Additionally,the time scales associated with the thermal reversions are on theorder of seconds for the dark thermal reversion of Pfr to Pr and onthe order of hours for the thermal reversion from Pr to Pfr (Section S6). This observation suggests that theprobability of sampling the thermal reversion in canonical phytochromesis indeed higher for the Pfr to Pr transition. However, for bathyphytochromes, this behavior may be reversed as for such phytochromesthe Pfr form is the main resting conformation.61Aspreviously noted, the crystallographic state of the Pfr form , is propagated uphill  that, due to the limitationof our approach, cannot be detected here and lands at the Pfr FE basin(0 kJ/mol). In the reverse transition (Pfr to Pr), the system startsdynamics in the X-ray FE basin of Pfr (0 kJ/mol), crosses the FE barrierof around 75 kJ/mol, and lands, through Pfr transient conformations,in the Sh metastablestate of FE around 0 kJ/mol. In this case, the FE basins are of similardepths , therefore the changes in PHYoccur with a considerable delay. In Figure S12 for additional results.In Section S11). Byidentifying the most frequent residue–residue contacts, wecan trace where the changes in the structure occur and whether itis possible to link these conformational changes to the enhanced chromophoredynamics. In Figure S12, from these results,we can identify two distinct structural pathways, one proceeding throughthe partially disordered tongue region and the other associated withthe movements of the spine helix.Additionally, we analyze the occurrence frequency of residue–residueclose contacts  during the enhancedsampling simulations . Our results suggest that, for Pr, the averagenumber of H-bonds between the protein and the chromophore is abouttwice as large in comparison to Pfr. This indicates that in the Prconformer the BV chromophore is more constrained in the CBD. In contrast,BV in Pfr can fluctuate more freely . This corroborates our results from calculating the FE landscapes,as the Pfr conformer has FE basins that are wider in comparison toPr  values for the time-series of an anglequantifying the rotation for the Pr and Pfr forms. We define thisangle based on the CHB and CHC  of the BV chromophore and the Cαatom of Leu-235 lying in the plane of BV . We perform this estimation using bootstrapping.Our analysis yields angles of 2.45 ± 0.2 rad and 2.63 ±0.2 rad for Pr and Pfr, respectively. The BV chromophore exhibitsreorientations by about 0.2 rad in both Pr and Pfr states, and thereis almost a 0.2 rad change in orientation between two forms, althoughthe means agree within the margin of error. However, as we can seein Figure S7, the distributions of thein-plane angles are different in Pr and Pfr. Namely, the distributionfor Pfr is bimodal. The first peak corresponds to the rotations similarto that of Pr (∼2.5 rad) and the second cannot be observedin Pr (∼2.7 rad). The second peak in the distribution of thein-plane angle for Pfr is more populated, and hence, we can observethe propensity of the BV chromophore to assume a conformation differentfrom that in Pr.Some discussion is in order regarding to the flip-and-rotate model,Overall, our simulations suggests that theprimary photoisomerizationevent which causes the chromophore to change the rotation angle ofthe BV D-ring destabilizes the CBD around the D-ring replacing stableinteractions to form a loose pocket in which the H-bonds between theCBD and BV are far less frequent. The D-ring rotation makes changesin the disordered part of the tongue which subsequently leads to asecondary structure change of the tongue from an antiparallel β-sheetto an α-helix, but it is the disordered part of the tongue regionthat allows for that conformational change through direct interactionswith the chromophore. Despite the clear differences in the angle distributionof BV in the Pr and Pfr forms, it remains to be further studied ifthe in-plane rotation of the BV chromophore relative to the proteinmatrix is an important factor for the rearrangement in the CBD.444 these steps lead the complex to populate the Pfr conformer. Whatwe can see is how different is the conformational behavior of thechromophore within the protein matrix in the Pr and Pfr conformers.By using enhanced sampling MD, we push these conformers out of equilibriumstates that are not accessible thermally. Simulating these processesusing unbiased MD would only give us a fragment of the whole pictureas indicated by recent applications of enhanced sampling MD to photoactiveproteins.81 We plan to employ quantum calculations  for the photoisomerization process in further studies.We need to underlinethat, by design, we cannot see the ultrafastphotochemical steps during the photoisomerization process in our enhancedsampling MD simulations. However, regardless of the transition throughshort-lived intermediates,82 We cannot answer the questionif the Φ dihedral is the only required degree of freedom toquantitatively describe the BV-BphP photoisomerization in experimentalconditions. However, it is clear that by biasing both dihedral angles,we are able to provide a detailed thermodynamic characterization ofPr and Pfr. Recent studies have shown that photoisomerization events,initially assumed to alter only the double bond rotation, can modifyalso the single bond.83 From the FE landscapes, we can see that the transition pathwaysinvolving both dihedral angles are highly possible, at least in theground state FE landscapes.In our work, we discover that the Pr and Pfr conformers are characterizedby multiple metastable states. This fact can be uncovered as we monitorand bias both the Φ and Ψ dihedral angles. Conversely,not all metastable states can be observed just by looking at the Φdihedral angle. This indicates the importance of selecting a set ofCVs that can describe the studied systems while losing as little informationas possible.17 and the PAS-GAFhelical spine75 to the terminal part ofthe PHY domain. This observation indicated that the mechanical signalcould propagate by pathways found by Takala et al.17 via the X-ray structures and by Isaksson et al.75 using NMR.Another open question that cannotbe answered even by exploitingthe most advanced experimental techniques, is which structural changesof phytochromes are the most important to propagate the mechanicalsignal from the CBD to the PHY domain. Whereas it is possible to gaingeneral knowledge about the identity of structural rearrangementsfrom IR studies, the insight obtained is far away from an atomic-levelresolution. On the other hand, XFMS experiments, which have a single-residuespatial resolution, cannot provide time-resolved data at the requiredaccuracy level. Our model, on the contrary, provides a detailed thermodynamicand atomistic description of the additional driving force caused bythe enhancing the local fluctuations of the BV chromophore for themultiscale structural changes of the protein involving the Pr andPfr conformers. We find that the mechanical signal is possibly transferredthrough the tongue regionQ-band of absorptionof phytochromes lies within 660–700 nm,38 which corresponds to an energy range of around 155–181kJ/mol. As shown in this study, the estimated FE barriers are in accordwith the fact that the excitation energy must be higher to reach anexcited state, which converts Pr via the conical intersection to Pfr.We suggest that the energy dissipation in the protein may play a largerole65 in the difference between the Q-band energies and the FE barriers estimated using VES,as some energy in the form of heat is dissipated to a thermal bath.Notably, the FE barriers estimatedin this study agree qualitativelywith the absorption spectra of Pr and Pfr. We show that for the protein-chromophore,higher energy is required in the change from Pr to Pfr compared tothe reverse transition. The 84 which is indicated by the existence of temperature-dependentchromophore conformations in many phytochromes.35 The Pfr state of canonical phytochromes has been shownto be heterogeneous in some cases, with a temperature-dependent equilibriumsuggested to be important for the thermal reversion.34 In this study, we show that an interplay between two differentPfr chromophore conformations exists. These metastable states areseparated by a relatively low FE barrier that renders the transitionsbetween those states to be on the microsecond time scale (Section S6). This fact is also in agreement withthe study by Salewski et al.,60 where ithas been suggested that the Pfr conformer is more prone to populatingthe CBD with heterogeneous transient chromophore conformations.Perhaps the most important result of our study is a possibilityof the dark thermal reversion from Pfr to Pr. It has been shown experimentallythat the BV-like chromophore may adopt alternate conformations71 We also observe that, during the dark thermal reversion, the aminoacid triad of Arg-466, His-467, and Ser-468 rotates clockwise, breakingthe H-bond between Ser-468 and the D-ring pyrrole. This clockwiserotation seems to be biologically important in the stabilization ofthe Pr conformer via H-bond with Arg-466 and a closing motion of theCBD around the BV chromophore. This process provides information abouthow the protein environment affects the heterogeneous photodynamicsof the chromophore and the possible conversion between β-sheetto α-helix in the conserved tongue region of BphP.We show that the disordered part of the tongue region is heavilyaffected by the rotation of the D-ring pyrrole of BV. Similar behaviorhas been observed in ligand dissociation.Insummary, this work is a next step toward elucidating the completemechanism of the photoisomerization of a bilin-like chromophore inphytochromes from the thermodynamical point of view. Considering theagreement with existing experimental data and the consistency of ourresults, our findings may be important for the basic understandingof the conversion between photoproducts in light-responsive proteins.