Many filamentous fungi colonizing animal or plant tissue, waste matter, or soil must find optimal paths through the constraining geometries of their microenvironment. Imaging of live fungal growth in custom-built microfluidics structures revealed the intracellular mechanisms responsible for this remarkable efficiency. In meandering channels, the Spitzenkörper (an assembly of vesicles at the filament tip) acted like a natural gyroscope, conserving the directional memory of growth, while the fungal cytoskeleton organized along the shortest growth path. However, if an obstacle could not be negotiated, the directional memory was lost due to the disappearance of the Spitzenkörper gyroscope. This study can impact diverse environmental, industrial, and medical applications, from fungal pathogenicity in plants and animals to biology-inspired computation. Neurospora crassa in microfluidic environments to show how constraining geometries determine the intracellular processes responsible for fungal growth. We found that, if a hypha made contact with obstacles at acute angles, the Spitzenkörper (an assembly of vesicles) moved from the center of the apical dome closer to the obstacle, thus functioning as an internal gyroscope, which preserved the information regarding the initial growth direction. Additionally, the off-axis trajectory of the Spitzenkörper was tracked by microtubules exhibiting “cutting corner” patterns. By contrast, if a hypha made contact with an obstacle at near-orthogonal incidence, the directional memory was lost, due to the temporary collapse of the Spitzenkörper–microtubule system, followed by the formation of two “daughter” hyphae growing in opposite directions along the contour of the obstacle. Finally, a hypha passing a lateral opening in constraining channels continued to grow unperturbed, but a daughter hypha gradually branched into the opening and formed its own Spitzenkörper–microtubule system. These observations suggest that the Spitzenkörper–microtubule system is responsible for efficient space partitioning in microenvironments, but, in its absence during constraint-induced apical splitting and lateral branching, the directional memory is lost, and growth is driven solely by the isotropic turgor pressure. These results further our understanding of fungal growth in microenvironments relevant to environmental, industrial, and medical applications.Filamentous fungi that colonize microenvironments, such as animal or plant tissue or soil, must find optimal paths through their habitat, but the biological basis for negotiating growth in constrained environments is unknown. We used time-lapse live-cell imaging of  Filamentous fungi dwell in geometrically, mechanically, and materially heterogeneous habitats, such as animal or plant tissue , 2, decaExtensive studies have described the fundamental growth behavior of fungi: For example, hyphal directional growth –11, regu12Advanced fluorescence microscopy studies of fungal growth on nonconstraining open surfaces have revealed several intracellular processes that are essential for hyphal extension and branching , 17, 18.19253137422, allowing in vitro studies in more realistic conditions.Microfluidics devices, which have been used to study the behavior of individual bacterial –50, mamm55Pycnoporus cinnabarinus and Neurospora crassa demonstrated differences in behavior in constraining geometries compared with that on flat surfaces; in particular, fungi grown in a geometrically constrained environment had up to 10 times lower apical extension rates and distances between branches. Translation of the fungal space-searching process into a mathematical formalism  , as “internal” control, as well as on agar, as “external” control.Because the vast majority of reported fungal growth studies have been performed on open agar surfaces, the first step in our study was to establish that the “internal” control in our experiments  provided comparable growth conditions with those reported in the literature. Therefore, we performed experiments in closed PDMS microfluidic structures comprising separate chambers (SI Appendix) . First, the cross-sectional apical profiles of the hyphae were parabolic and symmetrical . Second, the Spitzenkörper was centered at the hyphal apex , with small periodic oscillations perpendicular to the growth direction (Movie S2). Third, the microtubules were longer and less aligned with the hyphal axis when further away from the hyphal apex (Movies S3 and S4). This is seen as a broadening of the distribution of the deviations of microtubule angles from the hyphal axis . Furthermore, the lateral distribution of microtubules indicated that, while they populated both cortical and central cytoplasmic regions (the entire width of the hypha), their density was higher in the cortical region  . The microtubules extended into the apical dome, displaying a characteristic microtubule-depleted zone in the distal central region that colocalized with the Spitzenkörper (Movie S3). Long-term imaging (5 to 10 min) showed that microtubules occasionally traversed the Spitzenkörper position and frequently terminated at the apical cell wall. The estimated microtubule polymerization rate was 26.4 ± 8.6 µm⋅s−1 (n = 412 measurements from 98 microtubules). Finally, long-term imaging showed that this organization along the hyphal axis is interrupted when microtubules passed a septum .The comparison of fungal growth on agar  and in closed/nonconstraining conditions  demonstrates that they elicit similar growth behavior . The central positions and sizes of the Spitzenkörper were also similar .The lateral branching behavior (branching at ∼45° with movement of microtubules into the daughter hypha) was also similar on agar and in closed/nonconstraining PDMS chambers  when colliding with PDMS walls at acute angles. We found that nestling dynamics (Movie S7) present three phases:1)Before encountering the wall: Similarly to experiments in nonconstraining geometries, the hyphal profile was symmetrical, with the Spitzenkörper located centrally at the apex and the microtubules distributed symmetrically. We consistently observed the absence of any anticipatory change in behavior even before an imminent contact, suggesting the absence of any sensing mechanism.2)SI Appendix, Fig. S14, Top). Second, the longitudinal hyphal cross-section shape lost its symmetry and became considerably skewed toward the wall. The hypha continued its progress in close contact with the wall, maintaining this skewed tip profile. Third, the Spitzenkörper markedly shifted away from its previously central apical location, toward the wall. This displacement persisted over distances at least longer than several hyphal diameters . Skewing of the apex during nestling was constant over time: That is, in nestling events in a sequence of up to 10 chambers. Fourth, microtubules tended to gather near the inside edge of the hyphal bend . The nestling behavior of the Spitzenkörper  also occurred when a hypha was able to circumnavigate a small immovable obstacle .Nestling: We observed four major changes in hyphal morphology upon encountering a wall. First, the growing hypha followed the constrained path imposed by the obstacle as it slid along the wall in the direction of least deviation (3)SI Appendix, Fig. S17) (Movie S7 presents the complete time series); and the microtubules recovered their symmetrical transversal distribution. Within the spatial range of observation , the accuracy in the recovery of the direction of hyphal growth did not diminish over time, having negotiated successive bends through the device, or with increasing distance from the initial branching point of that hypha .Return to nonconstrained growth: After overpassing the end of the wall, within a distance approximately equal to the hyphal diameter, the hypha quickly recovered its original growth direction. Additionally, the hypha resumed its symmetrical profile; the Spitzenkörper simultaneously returned to a central position . Repeated imaging (n = 37 events) provided evidence for a three-phase intracellular process :1)SI Appendix, Fig. S20A).Polarized approach, before encounter (“Approach” in 2)SI Appendix, Figs. S19C and S21A). Hyphal growth then continued quasiorthogonally to the polarization axis, resulting in lateral bulging in the apical region. Simultaneously, the microtubules depolymerized, and the filament ends receded rapidly from the apex . At 25 ± 13 s after the collision, the average distance between the obstacle and the microtubule receding end was 7.3 ± 3.7 µm. The Spitzenkörper shrank gradually but did not retract longitudinally from the apical dome . In stage 2 . Importantly, the disappearance of the Spitzenkörper also occurred if the hypha pressed and then penetrated a PDMS wall (Movie S9). The microtubules resumed their extension toward the apex, and, after 80 ± 36 s from the collision, their population appeared to be fully recovered in the hyphae . In stage 3, just before branching was initiated and when the hypha did not have a Spitzenkörper, the uniform apical extension continued laterally, following the constraining geometry. The microtubules again extended to the extreme apical cell walls and migrated from the parent hypha into the nascent bulges, ultimately resulting in an extension along the obstacle walls .From the moment of encounter to branching (“Collision” in 3)r = 0.65, P < 0.05) with the initial diameter of the parent hypha. The change in polarization pattern coincided with the nucleation of two smaller “daughter” Spitzenkörper structures—one for each new branch . Independent microtubule populations developed within each branch to conclude the branching process .Branching . Finally, Movie S11 presents a similar lateral branching due to the collision of a hypha with a corner that does not allow the formation of two branches.Additional evidence of the intracellular processes during the hit & split in more complex geometries is presented in To establish the underlying intramolecular mechanisms responsible for growth and branching in tightly constrained geometries, we imaged the evolution of the hyphae in channels with widths smaller than their diameter, without and with lateral opening, and in dead-end corners.N. crassa progressed in long, linear, tight channels without lateral exits (n = 14), the hyphae branched immediately upon cessation of the confinement: For example, at a channel opening into a larger volume (Movie S12), with both hyphae generating their own Spitzenkörper soon after exit . Importantly, the behavior manifested during nestling  was also present . Additionally, the microtubules exhibited the same pattern: That is, pressing against the wall opposite to the initial direction of growth .First, when n = 25), branching occurred almost immediately when passing this opening .Second, for hyphae growing in channels with lateral exits (n = 20 hyphae):1)SI Appendix, Fig. S29). The microtubules were oriented longitudinally within the parent hypha .Entry and apical growth in the channel (“Approach” in 2)SI Appendix, Fig. S30A1). The longitudinal orientation of the microtubules in the parent hypha was conserved , but eventually polarization occurred , followed by microtubule transfer from the parent into the developing branch . Approximately halfway through this process (∼70 s) .Formation of a proto-branch SI Appendix, Fig. S30 E and F1). Interestingly, features associated with directional memory appeared early: For example, the ability of microtubules to cut corners , despite the necessity of passing through centrally located septa .Aside from observing the mechanisms involved in branching, the visualization of hyphae growing in tightly constraining channels offered additional evidence regarding the structuring of the microtubule cytoskeleton following changes of the direction of growth, now obligated by the meandering geometries. Similarly to nestling, the pattern of microtubules preferentially distributed toward the wall opposing the direction of growth (“cutting corners” patterns) was also observed when hyphae navigated meandering channels with widths of 5 µm  are dissimilar to the natural habitats of filamentous fungi, environments that comprise constraining geometries, which are expected to interfere with the mechanisms of fungal growth being studied. Our previous studies on the growth of the filamentous fungi abarinus  and N. c. crassa , 62 in PN. crassa in nonconstraining PDMS-made environments was similar to that on agar, both observed by us and as reported in the literature. First, in our experiments, the hyphal profile was parabolic and symmetrical , as also previously demonstrated and comprehensively described mathematically  as described in early classical studies (Movie S2) were consistent with a previous report , and their accumulation toward the apical region correlates well with previous observations (−1) was consistent with previously reported results obtained for hyphal growth on agar . One possible explanation for this hitherto unknown phenomenon is that the pressure applied to the hyphal wall due to the mechanical contact with the obstacle results in an intracellular signal that triggers consolidation of the hyphal wall at the zone of contact. This process would require the off-axis positioning of the Spitzenkörper and pressure on the contact point between the hyphal wall and the obstacle  . Furthermore, the off-axis position of the Spitzenkörper creates a skewed microtubule cytoskeleton, which leads to the characteristic pattern of “cutting corners” . This effect is even more remarkable when considering that the microtubules must pass through narrow septa, which are centrally located on the median line of the hypha  .Our results in nonconstraining environments  confirmed previous observations that hyphal growth follows the positions adopted by the Spitzenkörper . Howevercorners” A—especiaovie S5) , 69. TheMore detailed experiments regarding the role of F-actin structures—actin rings, patches, and cables —which arN. crassa , the Spitzenkörper was not visible until 50 s after hitting the obstacle. Moreover, the decrease we observed in Spitzenkörper size, its subsequent disappearance, and the assembly of two new daughter Spitzenkörper centers away from the parent represent a typical sequence of events that also occurs naturally in apically branching fungi: For example, Sclerotinia sclerotiorum . Furthermore, if a hypha encountered a corner (Movie S11), the resulting budding branch was not initially populated with microtubules, suggesting that the association of microtubules with the apical cell wall is not a prerequisite for selecting a branching site, as has been observed for lateral branching in nonconstraining environments . Consequently, it is reasonable to assume that the contribution of actin to hit & split branching is minimal.The role of actin in hit & split branching, as with nestling, is yet to be established. However, as it was shown for two species of yeast  and for . crassa , actin iWe found that the lateral branching that occurs in tightly constraining microfluidic channels was only partly similar to lateral branching in nonconstraining conditions. At the beginning of lateral branching in nonconstraining geometries, we observed the association of cortical microtubules with the cell wall at the location of the developing lateral branch. Upon further extension, the microtubules gathered and bent considerably. The severed ends of microtubules then migrated into the branch and resumed polymerization. These observations are consistent with other studies of lateral branching on flat agar surfaces . ImportaSI Appendix, Figs. S29 and S30). Also N. crassa branched typically and almost immediately after an exit from a bottleneck (Movie S12)  . These oN. crassa strain , suggesting that the nucleation of the Spitzenkörper occurs after the initiation of branching, as opposed to lateral branching on open surfaces  presents various levels of containment to fungal growth, from tight-constraining in channels with widths smaller than the hyphal diameter (5 to 7 µm) to confined, but nonconstraining chambers (100 × 100 × 10 µm). The design of the microfluidic network allowed the investigation of fungal behavior as influenced by various levels of confinement and constraint .The microfluidic network  mat a his-3+::Pccg-1-Bml+sgfp+ mutant strain (henceforth “Neurospora crassa GFP”) [Fungal Genetics Stock Center (FGSC) no. 9519] was used for the study. The high level of nutrients was necessary to ensure the canceling of the (possible) chemotaxis-driven growth directionality. The FM4-64 dye (Invitrogen Ltd.) was used as a marker for Spitzenkörper.Live-cell imaging used an inverted laser-scanning microscope  with photomultiplier detectors. Fluorescence and bright-field time-lapse images were captured simultaneously and analyzed using image processing software .Control measurements for fungal growth in nonconstraining environments were performed on 1% wt/vol malt extract media using somatic hyphae at the edges of the colony. Hyphal growth rates were measured by tracking the position of the extreme hyphal apices in subsequent frames. Fungal growth was recorded for the period needed to observe hyphal behavior from the entry in, to the exit from, the microfluidic network of interest, which require ∼20 min for a straight 100-µm channel. Due to the more convoluted geometries and the presence of multiple hyphae, in many instances, the image recording lasted more than 1 h. To measure the rates of microtubule polymerization within the apical compartment and to distinguish this from motility, the positions of individual filament ends were tracked frame-by-frame, following a methodology reported previously .n data points, reported for each instance. Statistical analyses included all accumulated data from at least 20 separate experiments (unless otherwise stated).Statistica 7.1 (Statsoft Inc.) and GraphPad Prism 6.01 (GraphPad Software Inc.) were used for statistical analysis and correlation tests. Statistical analyses included calculating the mean and SD values of parameters measured: i.e., position, alignment with the hyphal axis, polymerization rate for microtubules, times before reappearance of the Spitzenkörper, and hyphal bulge dimensions, over the total number SI Appendix.A full account of the methods is presented in Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


The increasing knowledge of adropin, afamin, and neudesin and the regulation of glucose metabolism and insulin resistance allows for the assessment of the differences in their concentrations between the groups with varied duration of diabetes mellitus (DM).  Assessment of serum levels of adropin, afamin, and neudesin in children with type 1 diabetes, with respect to the disease duration. n = 68) were compared to the control group (n = 70). The diabetic group was divided into 4 subgroups: (I) newly diagnosed patients, after an episode of ketoacidosis (n = 14), (II) duration no longer than 5 years (n = 18), (III) 5 to 10 years (n = 27), and (IV) longer than 10 years (n = 9). Serum concentrations of adropin, afamin, and neudesin were assessed and compared between the groups of patients. The criterion for statistical significance was p < 0.05.  The study consisted of 138 patients aged 5–18 years (M 40.58%). Children with type 1 diabetes ( The concentrations of adropin and afamin across all subgroups were lower than that in the control group, while neudesin levels were higher in diabetic patients compared to the control group. The differences were statistically significant.  Adropin, afamin, and neudesin may play a major role in the regulation of glucose metabolism and have a significant potential as novel biomarkers to predict future metabolic disorders. However, further multicentre studies on a larger cohort of patients are necessary to specify the role of these substances in the course and treatment of type 1 diabetes. The increasing knowledge of the regulatory peptides allows for the assessment of their role in linking the process of food intake, nutritional status, and body growth, largely through the regulation of glucose metabolism and insulin resistance . One of Another peptide is afamin, a member of the human albumin gene family which also includes albumin, vitamin D-binding protein, and fetoprotein . This glThe third substance assessed in our paper is neudesin. Mouse neudesin mRNA expression is detected in various tissues including the brain, heart, lung, and kidney . In the Most studies assessing the relationship of adropin, afamin, and neudesin with glucose metabolism provide data obtained from research conducted on animal models, adults with type 2 diabetes, and women with gestational diabetes. There are only few studies concerning these relationships in children. The study objective was to evaluate the concentrations of the selected peptides in blood serum of children with type 1 diabetes, taking into account the disease duration.n = 14), (II) duration no longer than 5 years (n = 18), (III) 5 to 10 years (n = 27), and (IV) longer than 10 years (n = 9). Statistical analysis was based on anthropometric parameters such as age, height, and weight and laboratory tests (HbA1c) using standard methods. We analyzed medical records of the patients with type 1 diabetes (method of insulin therapy and other diseases). Moreover, we used ELISA to measure serum concentrations of adropin, afamin, and neudesin in the patients. Statistical analysis was performed using Statistica 13.0. The nonparametric U Mann–Whitney test was applied to compare quantitative variables without normal distribution.The study population consisted of 138 children aged 5–18 years , diabetic patients of Diabetology Outpatient Clinic and Department of Pediatrics, Endocrinology, Diabetology with Subdivision of Cardiology, Medical University of Bialystok. The population included a group of 68 children with type 1 diabetes (diagnosed by ISPAD criteria) as compared to the control group of 70 healthy children with a negative history of inflammatory, autoimmune diseases, or cancer. The diabetic group was divided into 4 subgroups regarding the duration of type 1 diabetes: (I) newly diagnosed patients, after an episode of ketoacidosis (μg/ml (subgroup I) to max. 95.44 μg/ml (subgroup III) vs. control group 116.91 μg/ml. The mean levels of adropin and afamin were found to fluctuate during the course of the disease, whereas the mean level of neudesin remained stable  level was the highest in subgroup I (11.69%) as compared to subgroups II, III, and IV due to the fact that it contained recently diagnosed cases of type 1 diabetes. The second highest mean level of Hb1Ac was observed in the group of children with the longest disease duration (9.06%). The mean HbA1c among children in subgroups II and III was comparable (7.67% vs. 8.58%) .p=0.000001).Patients treated with continuous subcutaneous insulin infusion (CSII) had higher mean levels of all the peptides and lower mean HbA1c levels than children treated with multiple daily injections of insulin (MDI) (p=0.0005) and had lower body weight (p=0.00542) and height (p=0.0398)  .p=0.007176) and had a higher BMI (p=0.033627) than diabetic children without any other disease.Moreover, we noted that diabetic patients with coexistent diseases were statistically younger . A significant positive correlation between afamin levels and BMI was observed in a study by Paragh et al. . HoweverThe results of the present analysis showed a negative correlation between serum concentrations of regulatory peptides and level of glycated hemoglobin (HbA1c). Similar relationships were observed in a study by Kratochvilova et al., conducted in a group of obese adults with type 2 diabetes . HoweverIn our study, patients treated with continuous subcutaneous insulin infusion (CSII) had significantly lower mean HbA1c levels than children treated with multiple daily injections (MDI) of insulin. Similar correlations were observed in studies conducted by Beck et al.  and RuizIn the present analysis, the levels of the regulatory peptides were higher in children with coexistent diseases, such as autoimmune thyroid diseases, dyslipidemia, celiac disease, obesity, metabolic disorders, and hypertension. In a study by Köninger et al., afamin concentrations were found to be significantly increased in patients with polycystic ovary syndrome (PCOS) in comparison with controls . KronenbInsulin resistance is an important factor that affects the clinical course and metabolic control in patients with both type 1 and type 2 diabetes. In children with type 1 diabetes, insulin resistance is higher in comparison to their healthy peers . SeveralTo sum up, it should be emphasized that regulatory peptides can modulate insulin sensitivity both in healthy individuals and people with autoimmune diseases such as type 1 diabetes. Their serum concentrations not only affect glucose utilization  but alsoBased on the data obtained from animal studies, adults with type 2 diabetes, and women with gestational diabetes, it seems that adropin, afamin, and neudesin play a major role in the regulation of glucose metabolism and insulin sensitivity. This regulatory peptides have a significant potential as novel biomarkers to predict future metabolic disorders. However, further multicentre studies on a larger cohort of patients are necessary to specify the role of these substances in the course and treatment of type 1 diabetes.The levels of adropin and afamin may be associated with the time of current type 1 diabetes and may change during its course. There is a need for more research connected with this subject.BMI and HbA1c can affect the levels of the regulatory peptides in the body.


The proximal humerus is one of the most common sites of primary or metastatic malignant tumors. Reconstruction of the shoulder after tumor resection is controversial and challenging. When intra-articular resection is performed, biological reconstruction  may be the first choice rather than prosthetic reconstruction.To observe the mid- to long-term effects of oncologic, reconstructive, and functional outcomes of two different biological reconstruction methods for resection of humerus osteosarcoma involving caput humeri.n = 7) and tumor bone inactivated and reimplantation  in our clinic between 2007 and 2017. Patients’ general information, resection and reconstruction techniques, oncological and functional outcomes, and complications were collected and evaluated. Different complications of implantations were compared and analyzed for the different biological methods.This was a retrospective study of 13 consecutive patients who underwent humeral reconstruction of osteosarcoma including caput humeri using osteoarticular allograft (p = 0.04); caput humeri absorption occurred in all seven allograft patients and three TBIR patients at an average of 3.10 years after surgery; severe graft bone resorption appeared in five TBIR patients and two allograft patients at an average of 2.57 years of follow-up.The study included ten males and three females with an average age of 19.15 years. The operation time was about 3.65 h with an average blood loss of 631 ml. The resection tumor bones were 13–45 cm (23.54 cm on average). The mean follow-up period was 5.27 years. The shoulder movement was 10–70°  in abduction, 0–30°  in flexion, and 0–20°  in extention at the last follow-up. The complications included fracture in four TBIR patients and two allograft patients with an average of 2.67 years postoperation. Fracture rate was higher and appeared time was earlier in TBIR patients than in allograft patients (Humerus biological reconstruction involving caput humeri was associated with a high complication rate and acceptable limb function in the mid to long term. New combined biological methods should be explored and adopted in the future. The proximal humerus is the third most common site of osteosarcoma and the second most common site of all bony sarcomas, with a predilection for metastatic disease –3. AmputOptimal methods for shoulder reconstruction after resection of the proximal humerus including caput humeri are controversial and challenging , especiaWhen proximal humerus tumor does not invade the joint capsule or into the articular cavity, intra-articular resection can be performed. Surgeons can obtain a wide margin by preserving the proximity of the neurovascular bundle, deltoid muscle, sufficient deltoid, rotator cuff tendon, and part of the joint capsule to restore glenohumeral joint mobility, especially abduction , 15. Of The purpose of this study was to observe the mid- to long-term oncologic, reconstructive, and functional outcomes of patients who underwent two biological reconstruction methods for humerus osteosarcoma involving caput humeri, namely osteoarticular allograft (OAA) and tumor bone inactivated and reimplantation (TBIR).There were 131 patients who had proximal humerus malignant bone tumors and presented to our ward between January 2007 and January 2017. We used the inclusion criteria of biological reconstruction including osteoarticular allograft or autograft for treatment of primary osteosarcoma of bone; absence of prior surgical treatments for the sarcoma; complete clinical, radiographic, and pathologic records; and minimum follow-up of 3 years from bone graft reconstruction. We also excluded patients who received discontinuity therapy and missed the follow-up. The final study group consisted of 13 consecutive patients with humerus osteosarcoma involving caput humeri  exercises of the elbow , wrist, and hand. The average duration of immobilization in a thoracobrachial cast was 6 weeks. After removal of the cast or brace, the arm was supported in a sling for an additional 1–3 months. At the same time, patients began active ROM exercises and physiotherapy. All patients received additional adjuvant chemotherapy and routine follow-up every 3 months for the first 2 years, every 6 months for the following 2 years, and every 12 months thereafter. The involved shoulder joint functional results were described according to the physical examination findings including abduction, flexion, and extension before and after surgery. Radiographs and magnetic resonance imaging of the operated limb and CT scans of lung were made at routine intervals.p < 0.05 was considered to indicate a statistically significant difference. SPSS software  was used. All the data collection and processing were carried out by junior doctors.We defined various failure modes as events. An event constituted unsuccessful biological reconstruction including common complications such as fracture, nonunion, bone resorption, infection, hardware failure, graft removal, or amputation. For radiographic evaluation, fracture was defined as discontinuity of the graft bone; caput humeri absorption (CHA), as partial or complete humerus head resorption in the epiphyseal region; graft bone resorption (GBR), cortical, or total graft bone loss of more than 3 cm without new bone formation; and nonunion, no evidence of radiographic bridging of the approximated ends between the graft and host bone at least 2 months apart at a minimum of 6 months after the operation. Complication in different reconstruction methods was compared using the nonparametric Mann-Whitney test. There were 10 males and 3 females who underwent biological reconstruction of the proximal humerus, and their average age at surgery was 19.15 (9–34) years. The operation time was 3.65 (2.5–5) h, and the blood loss was 631 (400–1000) ml. The mean follow-up period was 5.27 (3–10) years. The average resection length was 23.54 cm  in 13 cases. Distant metastases occurred in 4 cases, of which 1 was treated by resection of the lung metastatic lesions with no evidence of disease. Three other patients had multiple lung nodes; they received second-line chemotherapy and died 1 year later. Thus, there were 10 patients with disease-free survival at the last follow-up.p = 0.04). The location was at the metaphysis in three and at the diaphysis in three. The metaphyseal fractures occurred without the protection of plate in two cases. Fracture appeared at the distal of autograft host junction in one patient after removal of fixation . The caput humeri absorption happened at an average of 3.10 (1–5) years after the operation. All patients had no pain or shoulder subluxation with limited movement of shoulder joint, especially abduction, and received nonoperative treatment. Severe graft bone resorption (GBR) was another complication in auto/allograft, which occurred after an average of 2.57 (1–5) years of follow-up in seven (53.85%) patients  in the TBIR  in abduction, 20–70°  in flexion, and 10–40°  in extension before surgery, and 10–70°  in abduction, 0–30°  in flexion, and 0–20°  in extension at the last follow-up in these patients with no difference in the two reconstruction methods.n = 1), compared to 40% (n = 4) in the allograft prosthesis composite and 62% (n = 8) in the osteoarticular allograft group after caput humeri resection. The authors concluded that endoprosthesis should be the first choice in keeping the functional results and glenohumeral stability, but their patients have primary benign or malignant tumor or metastatic disease at an average of 44.8 (16–83) years. The defect length of proximal humerus was not clear.Malignant tumors of the proximal humerus are challenging problems, and numerous reconstructive methods have been described , 19, 20.In our study, the patients were 19.15 (9–34) years old with a bone defect of 23.54 (13–45) cm. Residual humerus in E1 and E2 region or children were not suitable for endoprosthetic reconstruction because of the small length of remaining bone and narrow medullary canal. There was an extremely high rate of complications including fracture, especially at the metaphyseal graft; caput humeri absorption; severe resorption of the graft; and nonunion. We observed caput humeri absorption in all allograft patients (100%) in 5 years. Higher rate of severe graft resorption and fracture happened earlier in TBIR patients than in OAA patients. Unlike bone graft failure in the lower limb, patients with severe complications, even though all the bone graft was absorbed  years postoperatively. As with fracture, bone resorption is related to problems with the incorporation of the dead massive graft bone into the living host bone. If the living host bone has poor osteoinductive capacity or insufficient fixation or blood supply of the graft bone, the bone resorption may occur rather than bone formation. The risk may be reduced by achieving rigid, long, and stable fixation and combining with vascularized autologous bone graft , 26. TheAlthough much more severe complications happened in our study, biological construction such as OAA and TBIR remains a viable option for limb-sparing tumor resections in certain patients with tumor involved in most of S5 or E1 and E2 area or with skeletally immature skeleton. Endoprostheses are not always available in these patients for residue and small medullary canal fixation and big intramedullary stems. Moreover, the cortical resorption at the implant-bone interface, both in cemented and uncemented stems, influences the amount of bone stock available for future revisions in long-term survivor patients .The limitation of this study includes the retrospective character, the inevitable small number of patients, the limited following time, the selection bias of patients, and the operation methods. Further study should be taken in this area, such as combined allograft and vascularized fibula graft, TBIR, or allograft with prosthesis and fixation, in promoting the clinical effect and survival rate of the biological construction in proximal humerus.Humerus biological reconstruction including caput humeri was associated with a high complication rate and acceptable limb function in the mid to long term. New combined biological methods should be explored and adopted in the future.


The aim of this study was to test the effect of adding chicken eggshell powder (CESP) to conventional glass-ionomer cement (GIC) on its mechanical properties, and fluoride and calcium release. CESP was added with proportions of 3% and 5% by weight to the powder component of conventional glass-ionomer cement. The specimens were categorized into group A: GIC without CESP; group B: GIC with 3% wt. CESP; and group C: GIC with 5% wt. CESP; there were 12 specimens in each group. Groups B and C showed higher compressive strength values compared to group A. However, microhardness scores were higher in group C compared to groups A and B. As for ion-release results, group B displayed the highest values of fluoride release followed by group C at both 7 and 30 days. Group C showed the highest amount of calcium release followed by both groups B and C at 7 days, while at 30 days, groups A and B showed higher calcium release compared to group C. The mechanical properties of conventional glass-ionomer restorative material were enhanced by the addition of CESP. Moreover, fluoride and calcium release were not compromised by adding CESP. Since its introduction in 1972, glass-ionomer cement (GIC) has been popular among clinicians due to its exclusive properties such as chemical adhesion to mineralized tissues, moisture insensitivity, and low coefficient of thermal expansion, which is close to that of tooth structures. Moreover, GIC has superior biocompatibility, fluoride release, and rechargeability, which impart its anticariogenic properties . When GIMany attempts have been made to enhance the mechanical properties of conventional GIC, such as the addition of resin  and the Chicken eggshell powder (CESP) is composed of 98.2% calcium carbonate, 0.9% magnesium, and 0.9% phosphate, approximately; thus, it is considered a rich source of mineral salts, mainly calcium carbonate. Eggshell calcium is probably considered the best natural source of calcium . For thi3 in order to impart mechanical reinforcement to polyethylene/polypropylene composites used in different industrial applications [Chicken eggshell powder (CESP) has also been used as a rich source of CaCOications ,17,18. TAn anhydrous glass-ionomer restorative material  was used. This powder contains a blend of alumino-silicate glass and polyacrylic acid, which was mixed with deionized water with a ratio of 1 scoop powder: 2 drops water according to the manufacturer’s instructions.CESP was attained by calcination following the protocol of World Property intellectual organization (WO/2004/105912: Method of producing egg shell powder) . This can = 12).Group A: GIC without CESP (n = 12).Group B: GIC with 3% wt. CESP added to the powder component (n = 12).Group C: GIC with 5% wt. CESP added to the powder component , and CESP was added to the powder component with proportions of 3% and 5% by weight. The specimens were categorized into 3 groups:Cylindrical samples were fabricated using PTFE (Polytetrafluoroethylene) cylindrical molds with 4-mm diameter and 6-mm height for a compressive strength test , and oth−1 until failure occurred [Compressive strength was measured using a Universal testing machine  with an across-head speed of 1 mm minoccurred . Compres® ITW Company, Norwood, MA, USA). Three indentations were carried out for each specimen at 25 g force for 30 s, and the average score of the three readings was recorded for each specimen [Microhardness was measured using a Vickers microhardness tester machine  at 239.9 nm. Results were calculated as the amount of fluoride or calcium release per unit surface area of the specimen (μg/mm2).Twelve cylindrical specimens (6 mm in diameter and 3 mm in height) per group were prepared using mountable split Teflon rings as previously described. Equal lengths of paraffin dental floss were incorporated into the specimens during setting to suspend the specimens in the deionized water. Specimens were weighed to ensure standardization within each group using a digital balance (±0.0001 g) , and their dimensions (diameter and thickness) were also measured using the digital micrometer. Specimens were stored at 37 °C and 100% relative humidity for 24 h. Each specimen was then stored in an individual tightly-closed polyethylene tube containing 10 mL distilled deionized water at 37 °C. At the time of fluoride and calcium ions measurement, each specimen was removed from its container, and the storage solution was collected for analysis. The discs were then blotted dry and placed in a new container with fresh 10 mL distilled deionized water, and storage was continued [Data from all tested groups were collected, tabulated, and statistically analyzed using SPSS .One-way ANOVA followed by Tukey’s post hoc test was used to investigate the effect of adding eggshell powder on the compressive strength of the restorative material. The results revealed that groups B (3% CESP) and C (5% CESP) showed significantly higher compressive strength values compared to group A (GI without CESP), which showed the lowest values . However, microhardness scores were significantly higher in group C (5% CESP) compared to groups A and B, with group A displaying the lowest microhardness values. t-test of the effect of time interval on fluoride release showed that only group B had a significantly lower value of fluoride release after 30 days. Groups A and C also showed less fluoride release at 30 days than at 7 days; however, the difference was not significant for either group  trailed with the lowest statistically significant values in both tests. These findings reflect somehow the consistent enhancing effect of adding CESP on the mechanical properties of GIC. Since the mechanical properties of GIC improve with time, testing this novel material for extended time frames would have added extra value to the current research; however, the chosen testing time (24 h) only aimed at obtaining preliminary data to be considered as a cornerstone for future investigations.These results are in accordance with those of Lubis et al. , who alsSimilarly, Rahmi et al.  deduced The bioactivity of glass-ionomer cements stems from their capability of releasing ions, not only fluoride but also calcium and phosphates. These ions become incorporated in the nearby tooth structure to enhance its resistance to acid attacks . For thiDeionized water was selected as a medium for the estimation of fluoride and calcium ions release from the tested glass-ionomer samples; since it is devoid of any ions, it is considered a more accurate medium than artificial saliva and other acidic media ,35,36.Our results clearly showed that adding CESP did not interfere with the inherent ability of GIC to release ions, on the contrary, it even seemed to potentiate this ability to the extent that fluoride and calcium release were increased in groups B and C compared to GIC with no added CESP.Kumar et al.  obtainedSimilarly, Yli-urpo et al.  reported++ initially (before our first measurement), before it precipitated as CaP as reported in the previous study [++ values were depressed and came closer to the measurement obtained from group A (GI without any added CESP). At the same time, Ca++ in group C was not depleted by CaP precipitation, since samples in this group had a higher proportion of eggshell powder with a subsequent higher calcium content.The results of the current research, however, cannot be accurately compared to the aforementioned study, since our first measurement of calcium and fluoride was done at 7 days, followed by a second measurement at 30 days; this was done for the purpose of tracing long-term release of the ions that aid in remineralization. We detected the highest values of calcium release in group C, with CESP added to GIC in a proportion of 5% by weight, followed by group B, which had a slightly lower concentration of CESP (3% wt.); this could suggest that group B had also leached out too many Caus study , so thatWe can also postulate that the high calcium release in Group C (highest eggshell concentration) coupled with lower fluoride release could be explained by the ability of eggshell to absorb fluoride in solution, a theory which has been explained and utilized by various researchers ,40 in orThe mechanical properties of conventional GIC were enhanced by the addition of CESP.GIC unique property of fluoride release is not compromised by adding CESP to its powder component.Calcium release was potentiated at 5% CESP concentration, which can enhance the remineralizing ability of GIC.Within the limitations of the study, it can be concluded that:


This study aimed to investigate the differences in the serum levels of glucose, lipid, and thyroid function markers between unipolar and bipolar depressed patients, as well as the effect of anhedonia and suicidal thoughts on the levels of these biochemical parameters.A total of 287 unmedicated depressed patients from January 2016 to December 2017 were included in this study, including 92 bipolar depressions and 195 unipolar depressions. Anhedonia was determined using the item 32 of Symptom Checklist (SCL-90). Suicide ideation was assessed by item 15 of SCL-90.The bipolar group had significantly lower lipid levels  and insulin resistance index but higher levels of prolactin, low triiodothyronine (T3) and free T3 (FT3) as well as higher incidence of anhedonia as compared with the unipolar group. Depressed patients with anhedonia had significantly higher LDL level than those without anhedonia. Depressed patients with suicidal thoughts had cholesterol and high-density lipoprotein cholesterol (HDL) level. The above-mentioned differences were confirmed by logistic regression analysis. Receiver operating characteristic curve (ROC) analysis showed that the area under the ROC curve (AUC) ranged from 0.546 to 0.685.Triglycerides, cholesterol, LDL, VLDL T3, FT3 levels were significantly different between unipolar and bipolar depressed patients, which might have the potential to be the markers for differential diagnosis. Patients with anhedonia had lower LDL level, while patients with suicidal thoughts had higher levels of cholesterol and HDL as compared with the corresponding control groups. The link between serum lipid level and depression has been reported over the past three decades . The level of depression was evaluated by Self-rating Unipolar depression Scale of Zung (SDS) Z, . Unipola2(kg/cm2). Insulin resistance index was calculated by the formula Triglycerides/HDL , glucose, thyroid function parameters (thyroxine [T4]), free T4 [FT4], low triiodothyronine [T3], free T3 (FT3), thyroid-stimulating hormone [TSH]), prolactin and cortisol were collected from medical record. BMI = weight/heightno interest in things of 5 grades, and the score greater than or equal to 3 was defined as anhedonia. Suicide ideation was assessed by item 15 of Symptom Checklist-90 (SCL-90). It is a self-reported thought of death or dying of 5 grades, and the score greater than or equal to 3 was defined as suicide ideation.Anhedonia was determined using the item 32 of Symptom Checklist (SCL-90). It is a self-reported of feeling Continuous variables were presented as the mean ± standard deviation (SD) and were compared by Student’s independent t-test or one-way ANOVA depends on the number of groups. Fisher’s LSD test was used as a post-hoc comparison after ANOVA reaching significant. Categorical data were indicated by number and percentage (%), then were further compared by Chi-square test or Fisher’s exact test . Univariate (crude) and multivariate (adjusted) logistic regression were used to estimate the odds ratio (and its 95% CI) of independent variables to diagnosis or psychological index with dichotomous results . Point-biserial correlation coefficient was used between age (continuous) and dichotomous depression types. ROC analysis was used to investigate the diagnostic effectiveness of continuous variables to dichotomous results, and the AUC, sensitivity, and specificity were reported. All analyses were performed using IBM SPSS Version 20 .P < 0.05, Table A total of 376 subjects  were included in this study. There were 89 cases of healthy control (control group), 92 cases of bipolar depression (bipolar group) and 195 cases of unipolar depression (unipolar group). There were significant differences in the patient’s gender, age, and baseline SBP between the unipolar and bipolar groups . The FT4 level was significantly different in overall comparison (P < 0.05). Meanwhile, compared to the control group, the bipolar group had significantly lower levels of cholesterol, LDL, VLDL, T4 but higher levels of prolactin, FT3, FT4. The unipolar group had significantly lower T3 but higher cortisol level than the control group.The lipid levels and other biochemical parameters were compared among 3 groups  and between bipolar and unipolar groups. It was found that Triglycerides, Cholesterol, LDL, VLDL, prolactin, T3, FT3, and insulin resistance were significantly different in both overall comparisons and between two depression groups . The incidence of anhedonia was higher in the unipolar than in the bipolar group (P = 0.003).Participant’s psychological index including anhedonia and suicidal thoughts (both were assessed by SCL90) were compared. As shown in Table P = 0.047). The suicidal thoughts group had significantly higher levels of Cholesterol and HDL than the non-suicidal thoughts (both P < 0.05).To investigate the lipid levels and other biochemical parameters in the depressed patients with or without anhedonia/suicidal thoughts, subgroup analysis stratified by anhedonia and suicidal thoughts was performed. As shown in Table n = 287). As shown in Table P < 0.05). However, ROC analysis showed that the most AUCs ranged from 0.55 to 0.68, indicating medium diagnostic effectiveness.To further confirm the above findings of different levels of biochemical parameters between groups, logistic regression, and ROC analysis were performed in all depressed patients .Multivariate (adjusted) logistic regression adjusting for patient’s age, gender, triglyceridess, HDL and LDL cholesterol levels was conducted to investigate the results between independent variable and depression diagnosis (bipolar vs. unipolar depression). However, in multivariate results, age was the only variable significantly different between the bipolar and unipolar depressions Table . For theIn this study, we investigated the differences in the serum levels of glucose, lipid, and thyroid function markers between unipolar and bipolar depressed patients, as well as the effect of anhedonia and suicidal thoughts on the levels of these biochemical parameters. The results showed that the bipolar group had significantly lower lipid levels  and insulin resistance index but higher levels of T3 and FT3 as well as a higher incidence of anhedonia as compared with the unipolar group. Depressed patients with anhedonia had significantly higher LDL level than those without anhedonia. Depressed patients with suicidal thoughts had higher cholesterol and HDL levels. The differences in the serum lipid and T3, FT3 levels between the unipolar and bipolar groups were confirmed by Logistic regression analysis. The differences in LDL and cholesterol/HDL in subgroup analysis stratified by anhedonia and suicidal thoughts were also confirmed by Logistic regression analysis. ROC analysis showed that AUC ranged from 0.546 to 0.685. Taken together, these results suggested that triglycerides, cholesterol, LDL, VLDL T3, FT3 levels were significantly different between unipolar and bipolar depression.The AUC value of 0.5 to 0.7 means moderate diagnostic effectiveness, indicating the association between independent continuous variable and dichotomous outcome. However, an AUC value < 0.8 may imply insufficient diagnostic effectiveness in clinical practice. Small sample size and unexpected confounding sources also influence the estimation of AUC. Future works of larger sample size study and the control of confounding are needed to verify the effectiveness of these independent variables. The assessment of anhedonia and suicidal ideation by SCL-90 item 32 and 15 was based on previous studies . This phenomenon should be attributed to the fact that bipolar depressed patients have a younger age at illness onset than the unipolar depressed patients  occupied the most variance of explanatory variables, making other variables difficult to be significant in the multivariate logistic regression analysis. Therefore, the results of the multivariate model seemed to be not credible, and the results of univariate (crude) logistic regression and ROC analysis should be considered.We noticed a remarkable female preponderance  in our unipolar depressed patients. The gender difference in unipolar depression has been widely reported Kuehner, . At presP = 0.168). These results indicated that although the two forms of depressed patients had a comparable level of depression, there was still a difference in lipid profile between groups. However, the trend and types of these metabolic dysfunctions are not exactly consistent with a previous study  with anhedonia had significantly lower LDL level than those without anhedonia. Our result is inconsistent with Moreira et al. recent report that subjects with unipolar depression and anhedonia have a significant increase in LDL level Moreira, . The difOur subgroup analysis stratified by suicidal thoughts showed that depressed patients with suicidal thoughts had significantly higher cholesterol and HDL levels than those without suicidal thoughts. Our finding is similar to the previous study that recent suicide attempters had a lower serum triglycerides and a higher HDL than those without suicide attempt in depressed patients   and T3 and FT3 between unipolar and bipolar depression, which might have the potential to develop as the markers for distinguishing bipolar from unipolar depression. Patients with anhedonia had lower LDL level, while patients with suicidal thoughts had higher levels of cholesterol and HDL as compared with the corresponding control groups.


Worldwide olive industry has expanded into new climatic regions outside the Mediterranean basin due to an increase in extra virgin olive oil demand posing new challenges. This is the case of Uruguay, South America, where the olive crop area reached 10,000 hectares in the last 15 years and is intended to the production of EVOO. Uruguay has a temperate humid climate with mean precipitations above 1,100 mm per year but unequally distributed, mild winters, and warm summers, with mean annual temperatures of 17.7°C. Different agroecological conditions require local knowledge to achieve good productivity whereby the objective of this work was to show the feasibility and potential of olive oil production under our climatic conditions. For this the agronomic performance of Arbequina, Barnea, Frantoio, Leccino, Manzanilla de Sevilla, and Picual cultivars was evaluated along 10 years of full production. Phenology behavior, vegetative growth rate, productive efficiency, alternate bearing, and oil yield were determined. Sprouting and flowering processes occur in a wide window within the annual cycle between the months of August to November with great interannual variation. More than 8 t/ha fruit yield and 40% oil yields in dry weight basis were obtained in promising cultivars. However, alternate bearing arose as the main production limiting factor, with ABI values greater than 0.60 for most cultivars. We conclude that olive oil production in humid climate regions is feasible and the most promising cultivars based on productive efficiency are Arbequina and Picual. In the last decades, the worldwide olive industry had expanded into new climatic regions outside the Mediterranean basin due to an increase in extra virgin olive oil (EVOO) demand associated to its beneficial effects on human health . Among tOliviculture devoted to EVOO production is considered a new agricultural activity in Uruguay. However, olive introduction dates back to 1,780 along with the Spanish settlers  and the Modern olive plantations in Uruguay follow an intensive rainfed system within a range of 285–400 trees/ha, identified as system “S5” by IOC . Less thThere is extensive knowledge of the behavior of olive cultivars in traditional Mediterranean production areas, but not in regions with temperate-humid climate. Evaluation of a fruit species in a specific location requires the evaluation of its growth and development potential, precocity, and productive behavior . AlternaTo identify limiting factors in olive production is necessary to start with cultivars phenological studies. After several years, the average and the overlap flowering period can be identified among cultivars in relation to climatic characteristics . NegativThe objective of this work was to assess the feasibility and the potential of olive oil production in temperate-humid climate. For this, we evaluated the agronomic behavior of six cultivars characterizing their adaptability through phenology, growth rate, productive efficiency, alternate bearing index (ABI) and oil yield. This information is crucial for identifying promising cultivars in order to develop an integrated production strategy as well as to pursue technological development facing the expansion into new regions.Two experiments were established, one at INIA “Las Brujas” Experimental Station in Southern Uruguay  and the other at INIA “Salto Grande” Experimental Station in Northern Uruguay .At INIA “Las Brujas” Experimental Station (LB), olives trees of Arbequina, Barnea, Frantoio, Leccino, Manzanilla de Sevilla, and Picual cultivars were planted in 2002 at a density of 416 trees per hectare (4 m between trees and 6 m between rows). A randomized complete block design with four replicates and three trees per experimental unit was used. The soil at this site has a fine textured A horizon, with a maximum depth of 20 cm, with 2.5% of organic matter and pH 6.5 corresponding to a Typic-Vertic Argiudolls soil according to the USDA classification . The preAt INIA “Salto Grande” Experimental Station (SG) olive trees of Arbequina, Frantoio, Manzanilla de Sevilla, and Picual cultivars were planted in 2003 at a density of 333 trees per hectare (5 m × 6 m). A randomized complete block design with six replicates was used and three trees per experimental unit. The soil at this site has a coarse textured A horizon, with a very good natural drainage and a maximum depth of 50 cm; therefore, there is no need for raised beds even though the strong textural differentiation between soil horizons. Soil natural fertility is poor, with less than 1.8% organic matter and a pH of 5.5. This type of soil corresponds to a Udifluvent soil according to the USDA classification .Saissetia oleae and Acromyrmex and Atta ant species, respectively. For diseases, between four to six sprays per season with preventive cupric sprays complemented with other fungicides  were applied for controlling Olive scab (Venturia oleaginea), Cercospora leaf spot (Pseudocercospora cladosporioides) and Anthracnose olive rot (Colletotrichum spp.)  all throughout the irrigation season. Pest management was according to Integrate Pest Management guidelines  with conhttp://www.inia.uy/gras/Clima/Banco-datos-agroclimatico). Daily average mean temperature differed in almost 3°C between the two sites, with an average of 16.3°C and 19.0°C at LB and 19.0°C and 21.8°C at SG, for October and November, respectively. Also, the average effective precipitation was 109 and 105 mm per month in October and November at LB, and 134 and 142 mm at SG   from Mayf 12.5°C .Phenological records were made through visual assessments in individual plants based on the BBCH scale  along 10st. At INIA Salto Grande, only the flowering stages were recorded. Flowering length days number was calculated by date calendar subtraction between states 61 and 68 of the BBCH scale.Sprouting and flowering windows were defined based on the first and the last date registered for each stage per cultivar between 2007 and 2017, and the dates were registered as the day of the year (DOY), starting on January 13/tree) was measured only in the central tree of the plot during the first years (2007 to 2011) and in all trees of each plot from 2012 onwards. CV was calculated according to the ellipsoid volume method defined by three semi-axes (equation 1) with Eb and Ec as the North and East semi-axis of the tree, and Ea as the difference between tree height minus height of the first leaf and divided by two (Fruit production (kg/tree) was measured per tree at both sites  and then fruit yield (t/ha) was calculated combining fruit production (kg/tree) and tree density (trees/ha) data. Canopy volume  was estimated considering the years with high production (years “on”) and the ABI was calculated according to For the period 2010–2017 and only for LB site, the productive efficiency  was measured on a sample of 200 g of olives per cultivar, with three replicates . Each saOlives were collected only at “on” years  at fruit ripening index (FRI) 3 based on a 0–7 scale  with 0: i = number of olives with i ripening level, I = ripening level going from 0 to 7, N = total number of olives evaluated (100 olives).where n× site” was studied. To highlight the productive potential of the cultivars for each site, an independent analysis per site is presented. For canopy development data from 2007 to 2010 was analyzed by a non-lineal regression following an exponential model (equation 4) and goodness of fit was estimated by Pearson correlation between measured and predicted values.Most data were subjected to analysis of variance (ANOVA) and the interaction “cultivar p ≤ 0.05 were calculated to separate means. The statistical program used was InfoStat version 2017  were recorded along 10 years for six cultivars at LB and four at SG. Phenological data was analyzed following two different approaches: the lifespan in which the phenological phase occurred Figure 1Sprouting windows started between the first week of August and the second week of September Figure 1Flowering windows from 10% flowers open (BBCH 61) to most petals fallen (BBCH 68) was highly variable among years. Considering all cultivars, the earliest date for BBCH 61 was 268 DOY in Manzanilla de Sevilla and the last date for BBCH 68 was 320 DOY in Arbequina. Therefore, the window for flowering in Southern Uruguay was 52 days, from the end of September to the end of November, for the 10 years evaluated.The beginning of full bloom (BBCH 65) period at LB occurred between the second week of October and the second week of November, with a lifespan of 20 days in Barnea and 30 days in Manzanilla de Sevilla and Picual. At SG, the beginning of BBCH 65 occurred between the first and third week of October, with a lifespan of 5 days in Manzanilla de Sevilla and 17 days in Arbequina.The average flowering length varied between 11.3 and 15.1 days at LB for Leccino and Arbequina, respectively, and between 11.3 and 12.0 days at SG for Arbequina and Frantoio, respectively. Despite cultivar nor site, the shortest flowering length was 7 days and the longest was 29 Table 1.The flowering behavior in two contrasting years regarding its thermal regimes was different Table 2.3/tree) and Arbequina the smallest one (CV: 26 m3/tree) (The cultivars showed differences in canopy volume (CV), Frantoio presented the higher values and Arbequina the lower ones Figure 2b of the exponential equation (equation 4) , estimated by parameter ation 4) Figure 2p = 0.24) were found in CV/ha between LB and SG for each cultivar when the row space was filled, fruit yields were expressed in t/ha. Cumulative fruit yield presented significant differences among cultivars and regions (p < 0.0001) (Since no differences ( 0.0001) Figure 3Alternate bearing behavior between the years “on” (high fruit production) followed by years “off” (low fruit production) was present in all cultivars Figure 33 canopy) calculated for the “on” years, whereas Barnea had the lowest one , whereas it was significant for average fruit yield (p < 0.001). Oil content in dry weight basis (DWB) and in fresh weight basis (FWB) were the highest in Frantoio and the lowest in Manzanilla de Sevilla in both sites. The average fruit yield (t/ha) per year was higher in Picual, Arbequina, and Frantoio in the LB site, followed by Manzanilla de Sevilla and Leccino and being the lowest in Barnea cultivar. In the SG site, Arbequina and Picual showed the higher yields, followed by Manzanilla de Sevilla and the lowest was for Frantoio cultivar , Cercospora leaf spot (Pseudocercospora cladosporioides), and Anthracnose olive rot (Colletotrichum spp.), with anthracnose being the riskiest one due to its direct damage on fruits and oil quality   supported this work. We are grateful to David Bianchi, Richard Ashfield, and Jonathan Dávila for their collaboration in this project.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.


Gastrointestinal microbiota play an important role in animal host immunity, nutrient metabolism, and energy acquisition, and have therefore drawn increasing attentions. This study compared the diversity of the gut microbiota of both wild and captive bharals, which is an ungulate herbivore of caprid from the Qinghai-Tibet plateau.The sequencing of the V4-V5 region of the 16S rRNA gene via high-throughput sequencing technology showed that the dominant bacterial phyla are Firmicutes and Bacteroides both in wild and captive bharals. However, their abundance differed significantly between groups. Firmicutes were significantly higher in wild bharals, while Bacteroides were significantly higher in captive bharals. Different diets are likely a key influencing factor in the diversity and abundance of gut microbiota in bharals.Changes in diets affect the diversity of gut microbiota and the relative abundance of pathogenic bacteria, increasing the risk of diseases outbreak in captive bharals. The results of this study suggest that the structure and function of the gut microbiota should be regulated via dietary intervention, accurate provision of an individualized diet, and optimization of the functional network of gut microbiota and its interaction with the host. This will improve the ex situ protection of wild animals. Due to the use of modern molecular technology, the gut microbiota can be utilized as a signal hub that combines environmental inputs  with genetic and immune signals. All of this affect the host metabolism, immunity, and infection responses , and plaPseudois nayaur) is a member of the Artiodactyla, Bovidae, Caprinae, Pseudois, and a national second-class protected animal on the list of key wildlife protection in China [Uncia uncia) [The bharal  , 18, anda uncia) . At presa uncia) , 19, beha uncia) , and sysa uncia) , 22. Dura uncia) . TherefoThis study investigated the composition and structure of the gut microbiota of bharals. The results lead to a better understanding of the digestive mechanism, provide a theoretical basis for the monitoring of abnormal physiological status, control the occurrence of diseases, and optimize the energy conversion rate of the food. The findings provide an important theoretical basis for the research digestive physiology of captive bharals. The results provide information to improve the diet of animals as well as for diagnosis and treatment of intestinal diseases. Furthermore, new research directions are initiated for the development of intestinal microecological agents.A total of 8,217,442 high-quality reads were obtained after data quality control, and fecal samples were classified into 3,878 OTUs , 2,443 of which were in the captive group and 3,166 in the wild group.Rarefaction curves and rank abundance curves are commonly used to describe the diversity of samples within a group. The rarefaction curves directly reflect the rationality of the sequencing data volume, and indirectly reflect the species richness of samples, as shown in Fig. In order to show the relative abundance of bacteria communities more intuitively, we selected the top 10 taxa and generated the relative abundance superposition histogram at phylum and genus level respectively in Fig. P < 0.01) than that of captive bharals (49.68%). The abundance of Bacteroides (40.98%) was significantly higher (P < 0.01) in captive bharals than in wild bharals (31.35%). In wild and captive groups, the main genera are Bacteroides and Alistipes, and the abundances of Bacteroides and Alistipes in captive bharals were significantly higher (P < 0.05) than that in wild bharals.At the phylum level, Firmicutes and Bacteroidetes are the dominant phyla of both wild and captive groups; however, the abundance of Firmicutes (60.35%) in wild bharals was significantly higher .The goods coverage index exceeded 99%, indicating a high level of diversity coverage in the samples. The observed species, Shannon index, and Simpson index in the wild group were significantly higher than in the captive group (P = 0.001) was found between wild and captive groups.To further analyze whether significant differences affected the microbiome structure between the wild and captive group, the multi response permutation procedure (MRPP) significance analysis method was used. The result was A = 0.1153 > 0, indicating that differences between groups were higher than within groups; consequently, the applied study grouping was reasonable and a significant difference  Fig. a and priUsing the LDA  Effect Size (LEfSe) analysis method, species with significant differences between groups were selected. The results included three parts: a LDA value distribution histogram, an evolutionary branch diagram (phylogenetic distribution), and an abundance comparison diagram of biomarkers with statistical differences (LDA SCORE > 4) between groups  between groups. In ruminants, Firmicutes play an important role in fiber and cellulose degradation, and can degrade cellulose into volatile fatty acids, which can be used by the host [Arrhenatherum elatius), carrot (Daucus carota), and artificial fodder. The utilized recipes are simple in structure; however, the protein, polysaccharide, and fat contents of the artificial fodder are relatively high. This might be the reason for the higher abundance of Bacteroides in the intestinal flora of captive bharals compared to wild bharals.At the phylum level, the core microflora of the gut microbiota consisted of Firmicutes and Bacteroides in both captive and wild bharals, accounting for more than 90% of the total gut microbiota. This result coincided with previous studies of the gut microbiota in other ruminants –27. Howethe host . Firmicuthe host . Althougthe host , 31, andthe host . The dieLEfSe analysis showed a number of bacteria that play an important role in metabolism of nutrients and the health of the host. Previous studies have reported that Alistipes shows a correlation with metabolites such as short-chain fatty acids, oligosaccharides, and amino acids. Lachnospiraceae and Ruminococcaceae belong to butyrate-producing bacteria. These bacteria and metabolites exert an important impact on host health , 34.In addition, the Alpha-diversity of the captive group was significantly lower than that of the wild group. The food sources accessible to wild animals are more diverse, thus providing bharals with many different types of nutrition, which may require a more diversified gut microbiota to help bharals complete digestion and utilization of these nutrients , 36.P < 0.01), Acidobacteria (P < 0.01) and Gemmatimonadetes (P < 0.01) in the captive group were significantly higher than in the wild group; these bacteria showed a certain pathogenicity [Further research has shown that many chronic diseases are caused by a decrease in the diversity of gut microbiota, including inflammatory gut disease and diabetes . Comparigenicity , 39. Howgenicity . These rThis relationship between gut microbiota and host health, and in particular, the impact of microbial dissonance on host health, indicates the importance of feasible strategies to optimize the gut microbiota via diet. This means that diseases caused by changes in the gut microbiome can be regulated through the diet of the host. Food types can be reasonably chosen, a number of nutrients can be appropriately supplemented, and the healthy development of gut microbiota can be promoted. This will be conducive to the protection of wildlife ex situ.This study described and compared the gut microbiota of wild and captive bharals and significant differences were found between both groups. The difference in diet may have caused a decrease of gut microbiotic diversity and an increase of relative abundance of pathogenic bacteria. The result would be an increased susceptibility to diseases in captive bharals, which is extremely unfavorable for their protection. These findings provide further research directions for the study of the effect of gut microbiota on the growth and development of bharals. Moreover, this study provides theoretical guidance for the diagnosis and detection of intestinal diseases in bharals, and can also be used as a reasonable and balanced reference for the diet of captive bharals. The findings provide a theoretical basis for the screening of probiotics and development of intestinal microecological agents.The difficulties of wildlife sampling and the religious beliefs of ethnic minorities are considered, feces samples were selected as test material in this study. At present, most of the research on wildlife in this aspect takes fecal samples as research objects, the results describe the structure of the gut microbiota of bharals. Therefore, during January 2018, a total of 240 fresh feces samples of wild bharals were collected from different regions near the Donggeicuona Lake in Maduo County, Qing Hai, China. The average temperature of Maduo in January is − 16.8 °C, which retained the freshness of the feces of wild bharals as much as possible. The collected samples were temporarily stored in an in-vehicle refrigerator (− 20 °C).Forty-four fresh feces samples were collected from 11 bharals in captivity at the Qinghai-Tibet Plateau Wildlife Zoo during December 2017. To prevent sample contamination, the bharals enclosure was cleaned in advance and none of the sample donors received antibiotic or probiotic therapy for the past three months. The collected samples were temporarily stored in dry ice (− 50 °C). All feces samples were eventually frozen and stored at − 80 °C for further analyses.® Stool Mini Kit . DNA concentration and purity were detected with 1% agarose gel. Diluted the DNA to 1 ng/μL with sterile water.Total genome DNA of samples was extracted by CTAB method using QIAamp® High-Fidelity PCR Master Mix (New England Biolabs), 3 μL (2 μM) forward and reverse primers, 10 μL (10 ng) template DNA and 2 μL ddH2O. The thermal cycling consisted of pre-denatured at 98 °C for 1 min, followed by 30 cycles, including denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s. Finally, 72 °C for 5 min.16S rRNA genes of distinct regions (V4-V5) were amplified used specific primer  with the barcode . All PCRThe 1 × loading buffer (contained SYB green) of the same volume was mixed with the PCR products and detected by 2% agarose gel electrophoresis. PCR products was mixed in equidensity ratios. Then, mixture PCR products was purified with GeneJET™ Gel Extraction Kit (Thermo Scientific).Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermo Scientific). The library was sequenced on an Ion S5™ XL platform to produce 400 bp/600 bp single-end reads.http://cutadapt.readthedocs.io/en/stable/). By using UCHIME algorithm  [http://drive5.com/uparse/) [Get single-end reads and filtered the quality of the raw reads to obtain high-quality clean reads   , the reago.html) . Then thuparse/) . Sequencuparse/) , 46.Qiime software (Version 1.9.1) was used to analyze the differences of alpha diversity indexes between groups. R software (Version 2.15.3) was used to plot rarefaction curves, rank abundance curves, and stacked histograms of the relative abundance. Ade4 and ggplot2 packages in R were used for PCA and PCoA analyses, respectively. LEfSe software (LEfSe 1.0) was used for LEfSe analysis, which defaulted to a filter value of 4 for the LDA Score. For MRPP analysis, the MRPP function of the vegan package for R was used.


Eco-friendly cellulose nanocrystal/silver/alginate (CNC/Ag/Alg) bionanocomposite films were successfully prepared by blending of CNC with Ag/Alg solution. The CNC was fabricated from cellulose microcrystal (CMC) by acid hydrolysis method. The Ag nanoparticles (AgNPs) were generated by using Alg as a reducing agent through hydrothermal process. AgNPs-included composite films showed characteristic plasmonic effect of the AgNPs with the maximum absorption at 491 nm and they also showed high ultraviolet (UV) barrier properties. The CNC/Ag/Alg composite films were analyzed by using scanning electron microscopy, transmission electron microscopy, optical microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction technique. Depending on the type of nanofillers, tensile strength of the composite films increased by 39–57% and water vapor permeation decreased by 17–36% compared with those of the neat Alg films. The Ag/Alg and CNC/Ag/Alg films showed brown color as detected from the increase of both ‘b’ and ‘a’ parameters by colorimeter. The UV and water barrier properties of Alg based composite films were found higher than the Alg films. The obtained results suggested that the prepared composite films can be used in food packaging applications. Plastic packaging films including poly(vinyl chloride), polyethylene, and polypropylene are widely utilized in the market owing to many advantages, such as low price, light weight, good mechanical performance, and water resistance. However, such films cause waste disposal problems in environment due to its non-biodegradable nature . So, tod3 solution. This composite showed improvement in mechanical, antibacterial, and water swelling ability of pure BC. To the best of our knowledge, the synergistic effects of incorporating CNC and Ag on the mechanical and barrier (water and ultraviolet (UV)) properties of Alg composite film has not been yet reported.Cellulose is the most abundant natural biopolymer on earth, occurring in wood and cell wall of plants ,24. It i+ by applying a “green” approach through hydrothermal method of using Alg itself as reductant. Afterwards, the biosynthesized Ag/Alg composite nanoparticles were mixed with CNC to fabricate CNC/Ag/Alg composite films using solution mixing then followed by casting technique. Further, the morphology, interactions among the components, and properties, such as color, mechanical, and barrier (water and UV) of the CNC/Ag/Alg bionanocomposite films were characterized by various techniques.The aim of this study was to fabricate Ag/Alg composite nanoparticles through chemical reduction of Ag® UFC100) was received from . Jardino universal potting soil (garden soil) was used for degradation test of fabricated films. Phosphotungstic acid, as staining agent, was received from Sigma-Aldrich. The deionized/millipore water was used in the whole research.Silver nitrate and sodium alginate were obtained from Sigma-Aldrich . Sulfuric acid was purchased from J.T. Baker, Taiwan. CMC (ArbocelThe CNC was fabricated from CMC using sulfuric acid hydrolysis method. The details for CNC fabrication was reported in recently published paper . The obtThe Alg solution was fabricated by dissolving Alg (0.95 g) in 80 mL deionized water with magnetic stirring for 1 h. Water dispersed CNC (by ultrasonication) was charged into the Alg solution to achieve CNC/Alg solution under stirring for 1 h at 27 °C. Afterwards, the CNC/Alg solution was spread on petri dish for film forming. The resulting films were treated with air drying process at 40 °C for 24 h to remove water. The neat Alg film was prepared according to the above mentioned procedure, except without CNC addition.3 solution and the above Alg solution (1 wt.%). The solution was further put in an oven at 60 °C for 12 h without any stirring. Next, the obtained brown solution of Ag/Alg was kept for magnetic stirring for 12 h. By a similar casting method to the CNC/Alg film preparation, the Ag/Alg composite film was prepared.The Ag/Alg solution was fabricated by mixing of 100 μL of 1 M AgNOThe Ag/Alg solution was mixed with CNC solution under stirring for 2 h. The resulting solution was casted on a petri dish to prepare CNC/Ag/Alg composite films. The details about the procedure for fabricating the Alg, CNC/Alg, Ag/Alg, and CNC/Ag/Alg solutions and films are shown in −1) using Bruker Tensor 27 IR spectrometer .The interactions among the components in fabricated composite films were analyzed with FTIR  was used for observing the surface and cross-sectional morphology of the prepared composite samples. The Energy-dispersive X-ray spectroscopy(EDS) analysis was carried out to confirm the presence of AgNPs in composite films.Carbon film covered copper grids  were used for imaging CNC samples. One drop of the CNC suspension (0.1 mg/mL) was placed on a grid for 30 min and wicked away with a filter paper. Further, the sample was stained by depositing a drop of (2 wt.%) phosphotungstic acid (PTA) solution on the grid for 20 min and wicking the excess solution away with a wet filter paper. The grid was put into oven at 70 °C for drying before insertion into the microscope. Finally, the CNCs were analyzed by a JEOL  operated at an accelerating voltage of 200 kV.Morphology and dispersion of CNC within (or without) Alg film were examined by OM .002 is the intensity of the crystalline region of cellulose (2θ = 22.5°), and Iam is the intensity of the amorphous region (2θ = 16.3°).XRD patterns of all samples were recorded with X-ray diffractometer . The crystallinity of CMC and CNC samples was calculated using Equation (1):−1.The degradation behavior and thermal stability of prepared samples were studied in nitrogen atmosphere by operating a thermogravimetric analyzer . The samples were heated from 40 °C to 700 °C at a rate of 10 °C min600 is the absorbance at 600 nm, and d is the film thickness (mm). The transparency of the films was determined following the light transmittance in the light wavelength region (ranged from 190 nm to 900 nm). The used scan rate was 1000 nm/min, and three replicates of each material were measured.The opacity of prepared films was defined by the following Equation (2) using a UV spectrophotometer  for the measurement:i and Wf represent initial and final dry weight of film.The procedure detail of FWS was followed by Yadav et al. . The fol2 dried at 105 °C for 24 h. The dried films were conditioned in a humid chamber  at 25 °C to attain 95% relative humidity. After a certain time, the films were removed from the chamber and weighed. The following formula [t and Wi represent the film weight at time t and before exposure to 95% RH, respectively.ASTM D5229  was foll formula  was used2), t is the time (s), and Δp is the vapor pressure difference.According to ASTM-E96/E96-05, the WVP of prepared films was studied. The details of the procedure was described previously . In eachThe color properties of the films were measured by using a TES-135A colorimeter (Taiwan). The obtained parameters are the lightness (L) and chromatic parameters: a (green (–a) to red (+a)) and b (blue (–b) to yellow (+b)) colors. Based on the measured parameters, the total color difference (ΔE) was calculated using Equation (6):The tensile properties of film specimens (10 mm × 60 mm × 0.01 mm) were determined (according to ASTM D638) using a Gotech AI-3000 system . A crosshead speed of 10 mm/min and, initial grip distance of 40 mm were used for the tests. The pull tests of the films in terms of tensile strength and elongation at break (%) was calculated by following formula:i). A total of 35 g of soil was poured into a plastic pot up to a thickness of approximately 1 cm. Then, the films were buried under soil at a depth of 0.5 cm from the soil surface. The assay was completed at 25 °C and (30%) RH by using a humid chamber . Films were taken from the soil at different times and cleaned carefully with tissue paper. Consequently, the films were dried in an oven at 105 °C for 6 h and weighed (Wt) to determine the percentage of weight loss using the following equation:Soil burial tests for biodegradation of the films were conducted by a method described by Martucci et al. . The fil−1 in CNC spectrum confirmed that CMC was acidified. In the Alg spectrum, the characteristic peaks at 1606 (asymmetric stretching vibrations of carboxylate anions), 1414 (symmetric stretching vibrations of carboxylate anions), and 3409 cm−1 (O–H stretching vibration) [3 was added to Alg, the peaks at 1606–1597 and 1414–1384 sharpened, thereby suggesting that AgNPs were generated by the reduction of Ag+ with Alg [−1 (in Alg) to 3426 cm−1 (in Ag/Alg film) suggests a certain degree of interaction occurred between Ag and O. The functional groups (–OH and –COOH) of Alg should be involved for fabricating AgNPs [−1 sharpened, thereby proving the miscibility (due to presence of hydrogen bonding) [−1 sharpened, confirming again that hydrogen bonding type interaction occurred between Ag/Alg and CNC.FTIR spectra of CMC, CNC, Alg, Ag/Alg, CNC/Alg, and CNC/Ag/Alg samples are shown in bration)  were assbonding)  between 3 was immersed in Alg solution, Ag+ could easily diffuse into Alg fibers and reacted with the fibers via ion exchange with sodium ion in the fibers due to the negatively charged Alg facilitating the attraction of the positively charged Ag ions by electrostatic attraction [+ ions decreased in situ, and the formed AgNPs closely deposited in the Alg fibers. Moreover, fine dispersion was observed, indicating that the in situ formed AgNPs could be effectively stabilized by the Alg fibers. The TEM images of fabricated CNC and AgNPs are presented in traction . Then, ni and Tf are the initial and final experimental temperature, respectively. A presentation of S1, S2, and S3 for calculating A* and K* are provided in a published research paper [max values of 54, 238, and 393 °C identified in the three-step degradation  provides an information about the water transfer from the food to its environment. The WVP of Alg, Ag/Alg, CNC/Alg, and CNC/Ag/Alg are presented in The thickness of packaging films generally depend on the three factors : structuThe physical appearance of the food product affects the consumer preferences. Therefore, the color of the packaging films directed a great effect in food industry. The obtained color variables L, a, b, and ΔE are presented in The ability of the food packaging films to block the UV light is an important attribute for food products. Thus, the transmission of ultraviolet (200–400 nm) and visible light (400–700 nm) through food packaging films played an important role for tracing proper package. The biopolymer based transparent food packaging films secure the pictorial confirmation of food content, which is an important reason that affects the consumer’s sense of food quality. Opacity is a key factor that controls the quality of packaging films. As presented in The optical properties of Alg, Ag/Alg, CNC/Alg, and CNC/Ag/Alg observed by UV−Vis absorption spectrophotometry are shown in Although the Ag/Alg, CNC/Alg, and CNC/Ag/Alg composite films showed lower transparent results compared to neat Alg film which is good feature for packaging materials. The light transmittance at different wavelengths of Ag/Alg, CNC/Alg, and CNC/Ag/Alg bionanocomposite in the UV region was presented in Biodegradability test is a highly important tool to recognize the environmental compatibility of materials. The biodegradability of food materials in the soil is already reported by many research group ,74. SpecIn this article, the needle-shaped CNC was prepared from CMC using acid hydrolysis and was used as reinforcement materials in bionanocomposites on the basis of Ag/Alg mixture. The solution blend technique was applied for fabricating CNC/Ag/Alg composite films. FTIR revealed that CNC and AgNPs interacted with Alg through hydrogen bonding, and good miscibility was developed among Alg, CNC, and Ag. The AgNPs-included composite films showed a specific plasmonic effect on the AgNPs with maximum light absorption at 491 nm and all the composite films, especially composed of AgNPs, showed the UV barrier property. The improved UV barrier property of the composite film can be used as a UV-screening food packaging film. Mechanical strength (determined by the TS), water barrier property, and thermal stability  of all the nanocomposite films increased significantly. The biodegradability of Alg and CNC/Alg films was found higher than other samples due to the absence of Ag metal. Based on the obtained results, the synthesized biodegradable nanocomposite films have the potential to be used as a food packaging material by using their barrier (water and UV) properties.