The number of additional airway interventions was the primary outcome. Percentage of patients with no need of additional interventions and apnea-hypopnea index (AHI) were secondary outcomes. The number of additional interventions was significantly less in the JED group compared to the conventional group (0 (0–0) vs. 1 (0–2); p = 0.002). The percentage of patients with no requirement for additional interventions was significantly higher in the JED group compared to the conventional group . AHI was significantly lower in the JED group compared to the conventional group (4.5 (1.5–11.9) vs. 9.3 (3.8–21.9), p = 0.015). In conclusion, JED seems to be effective in opening the airway patency during sedation in CRF patients.Patients with chronic renal failure (CRF) are likely to have obstructive sleep apnea (OSA) underdiagnosed, and maintaining airway patency is important during sedation. This study compared Jaw elevation device (JED) with conventional airway interventions  during sedation and hypothesized that JED may be effective to open the airway. A total of 73 patients were allocated to a conventional group ( Patients with chronic renal failure (CRF) are known to show a high prevalence of obstructive sleep apnea (OSA), which is often under recognized before surgery ,10,11,12Common clinical practice for securing the airway includes head tilt-chin lift, jaw thrust, head lateral rotation and oral airway insertion during sedation ,20,21,22Jaw elevation device  is a FDA (Food and drug administrated) approved, commercially available, externally placed, noninvasive device that assists anesthesiologists to maintain airway patency by lifting the mandible with an artificial jaw thrust arm. This device can be applied to sedated patients in supine position. A recent case report stated that JED is a practical device for reliable ventilation during neuroangiography and thrombectomy under sedation, and it was also effective after intervention for neck immobilization and airway opening .We hypothesized that with an aid of JED, we would reduce the number of additional airway interventions required during sedation in CKD patients compared to the conventional airway intervention maneuvers. Portable polysomnography (PSG) was also performed during sedation to assess for the differences in the degree of apnea-hypopnea index between the JED and conventional airway interventions. The purpose of this study was to determine the usefulness of the JED as an alternative option for maintaining the airway during sedation in CKD patients undergoing AVF surgery.2? (6) Age > 50? (7) Neck circumference > 40 cm? (8) Gender: male?This randomized controlled study was conducted at the Samsung Medical Center in South Korea between June 2017 and December 2020. All patients provided written informed consent. Adults aged above 20 years diagnosed with CRF, who were scheduled to receive AVF surgery under sedation with propofol and remifentanil between July 2017 and December 2020 were enrolled. Exclusion criteria were allergy to propofol, remifentanil or midazolam; history of central apnea; chronic use of antidepressants, opioids or analgesics; severe respiratory diseases; inability to extend or rotate neck; neurologic dysfunction; and temporomandibular joint dysfunction. Randomization was undertaken with a computer generated randomization table, and patients were allocated to apply the conventional airway interventions or Jaw elevation device . JED consists of a head support with memory foam and a jaw elevating apparatus at each side with a cushion at the surface touching the skin. All patients were asked to complete a STOP-BANG questionnaire preoperatively, which has been suggested by previous studies as an effective screening tool for sleep apnea ,26,27,282) sensor at the nares were recorded at the operating room. Type 3 portable polysomnography (PSG), Embletta MPR® , was applied at the patient’s nares  before induction, and 6 L/min of oxygen was supplied via facial mask. Facial mask was applied by an elastic bandage  at effect site concentrations (Ce) of 1.0–3.0 μg/mL for propofol, and 1.0–2.0 ng/mL for remifentanil. Sedation was adjusted to achieve the modified observer’s assessment sedation scale (MOASS) of 2–3. MOASS consists of 0–5; MOASS 0 was no response after painful trapezium squeeze, 1 was response only after painful trapezius squeeze, 2 was response only after mild prodding or shaking, 3 was response only after name is called loudly and/or repeatedly, 4 was lethargic response to name spoken in normal tone, and 5 was response readily to name spoken in normal tone.Anesthesia monitoring of electrocardiography, pulse oximetry, noninvasive blood pressure, heart rate, respiratory rate and sidestream end tidal carbon dioxide (ETCO bandage . Anesthe2 ≥ 8 s. The types of airway interventions were selected at the clinician’s discretion. Airway intervention types were as follows: (1) head lateral rotation, (2) neck extension, (3) oral airway insertion, (4) nasal airway insertion, (5) jaw thrust maneuver.At induction, baseline airway patency was confirmed by the attending anesthesiologist. Patients in conventional group were placed in a neutral position with a doughnut gel head pad . At PACU, patients were questioned about sedation satisfaction using a visual analogue scale  and the presence of pain at the jaw. We also inspected for color change at the jaw.Rescue interventions for in case of desaturation <93% during sedation was termination of the study and airway access with mask ventilation or supraglottic airway device insertion.2), and infused doses of remifenanil and propofol were recorded. PSG related parameters were retrieved from the Embletta MPR device using RemLogic™  polysomnography software. These parameters included analysis time, apnea-hypopnea index (AHI), total duration and percentage snoring time, and average SpO2. In PSG analysis, apnea was defined as breathing cessation lasting for 10 s or more, and hypopnea was defined as a fall of 30% or more of nasal respiratory flow signal. AHI was defined as the number of apneic and hypopneic events per hour of recording time. Snoring was recorded by a microphone assembled in the Embletta MPR device if 3 or more snoring was detected, and snoring periods were merged into one if the interval between them was less than 10 s.Intraoperative parameters including heart rate, mean blood pressure, respiratory rate, pulse oximeter  vs. 5–8 (high risk of OSA) was performed to compare the number of additional airway interventions and AHI between the groups.We expected that the number of airway interventions in the JED group would be reduced by at least 50% compared with the conventional group. Sample size was calculated so that at least 34 patients were needed in each group to detect a percentage difference in the number of airway interventions of 50% with a standard deviation of 2 with a power of 80%, α = 0.05. nQuery Advisor 4.0 software was used for the Mann–Whitney U test based on independent two sample t test for sample size calculation. Based on the estimated drop of 20%, recruitment was targeted to be 43 patients in each group. PSG data of a total of 6 patients (3 patients in each group) were missed because of a monitoring error. Therefore PSG data were analyzed excluding these missing data.p <0.05 was considered statistically significant.Statistical analysis was performed using SPSS statistics 21.0 . Data were presented as mean ± SD or median  for continuous variables and the number (%) for categorical variables. Continuous parameters were compared with t test or Mann–Whitney U test as appropriate. The normal distribution of the continuous variables was analyzed using the Shapiro–Wilk test. Categorical parameters were analyzed using the chi square test or Fisher’s exact test; Eighty-eight patients were assessed for eligibility, and 74 patients were finally enrolled. One patient in the conventional group was dropped because sedation was terminated early during surgery and the surgery proceeded with local anesthesia. Thus, 73 patients completed the study for the primary outcome. A CONSORT flow diagram is shown in Patient characteristics and preoperative survey data were similar between the groups .p = 0.002). The percentage of patients in each group with no requirement for additional airway interventions was significantly higher in the JED group compared to the conventional group . Among the types of airway interventions, head lateral rotation and neck extension were significantly higher in the conventional group compared to the JED group .Intraoperative sedation data and airway intervention parameters are shown in p = 0.015). Autonomic arousal index and relative snoring time were statistically not different between the groups. None of the patients complained of jaw pain after surgery, and they were all satisfied (VAS of 10) with anesthesia in both group. None of the patients showed desaturation.PSG data are shown in p = 0.003). AHI showed no statistical difference between the groups  vs. 8.2 (2.8–21.0); median difference, 4; 95% CI, 0–10; p = 0.075).Sensitivity analysis in patients with STOP-BANG of low to intermediate risk (score of 0–4) showed that the number of additional airway manipulation was significantly lower in the JED group compared to the conventional group (0 (0–0) vs. 1.0 (1–2.0); median difference, 1; 95% CI 0–1; Our results showed that with an application of JED during sedation, the number of additional airway interventions was less frequently required. Indeed, a significantly greater percentage of patients did not require additional airway interventions at all in the JED applied group compared to the conventional airway management applied group. We think it is a clinically useful finding that an externally applied, noninvasive device, JED, is favorable in airway management during sedation. AHI, a highly validated index for assessing OSA ,30, showInterestingly, sensitivity analysis showed that in patients with a STOP-BANG score of low to intermediate risk of OSA (score of 0–4), the JED group showed significantly fewer additional airway interventions compared to the conventional group. The patients in the two groups had shown a similar preoperative STOP-BANG score. JED seems to be effective in fairly low to intermediate risk of OSA patients in securing the airway. But in patients with higher risk of OSA (high STOP-BANG score), JED alone, which mainly elevates the mandible jaw to mechanically open the pharyngeal space, is not as effective as in patients with low to intermediate risk of OSA.The types of additional airways applied with JED were oral airway insertion or jaw thrust maneuver. In the conventional group, head lateral rotation, neck extension or oral airway insertion were the most frequently used airway interventions. We believe that, along with fewer airway interventions, JED can also free up the hands of the anesthesiologist manually maintaining jaw thrust or head turning and provide comfort to the patients. It could help the surgeons’ procedural convenience and reduce the chance of converting to general anesthesia with airway compromise during a procedure. The benefits of JED may render its use appropriate during procedural sedations in non-operating room anesthesia (NORA), the emergency department (ER) or intensive care unit (ICU).Portable PSG is a validated examination of recording of nasal airflow via nasal cannula and oxygen saturation via pulse oximetry ,31. In p2 were used to adjust the sedative agents to prevent severe respiratory depression.Limitations in our study include the followsing: First, preoperative PSG was not performed, instead we carried out a preoperative sleep apnea (STOP-BANG) questionnaire for all patients, and they were similar between the groups. The primary goal of the study was not to determine absolute sleep apnea status in CRF patients, but to compare the effect of JED during sedation. Second, we were not able to blind the clinicians to group allocation because either use of gel pad or JED was obviously seen. However, additional airway interventions were made by a clinician not involved in the study, and data analysis were also performed by a person not involved in the intervention. Third, sedation depth was monitored by a frontal electroencephalogram, and the BIS value ranges were quite wide. However, all patients were sedated according to the MOAAS at the discretion of clinicians. Fourth, the portable PSG measured the obstructive apnea, so we were not able to differentiate central apnea that may be associated with sedative drugs. During sedation monitoring, patients’ respiratory rate and sidestream ETCOIn conclusion, we were able to use less additional airway interventions by using JED, with lower AHI values during sedation. The externally applied jaw thrust device, JED, seems to be an effective aid to open airway patency compared to conventional airway manipulation during propofol and remifentanil sedation in CRF patients undergoing AVF surgery. With JED, both patient and clinicians benefit clinically with better safety and quality of sedation.


To report multimodal imaging of novel retinal findings in a case of syndactyly, telecanthus, anogenital, and renal malformations (STAR) syndrome.A 5-year old patient with STAR syndrome, an ultra-rare developmental disorder composed of syndactyly, telecanthus, anogenital, and renal malformations, was found to have bilateral macular yellow pigmentary changes and peripheral retinal pigment epithelial changes in a radial pattern highlighted by fundus autofluorescence (FAF) imaging. Optical coherence tomography (OCT) of the macula revealed foveal hypoplasia, ellipsoid zone disruption, and outer retinal atrophy suggestive of a retinal degeneration. OCT angiography found no significant abnormalities, and oral fluorescein angiography revealed staining in areas of atrophy in both eyes.This case displays the first report of multimodal imaging of retinal manifestations in STAR syndrome, revealing bilateral foveal hypoplasia, outer retinal macular atrophy, and peripheral retinal pigment epithelial changes. Further studies and long-term follow-up are warranted to determine if patients with STAR syndrome have an underlying progressive retinal degeneration. CDK10 plays an important role in cell division and proliferation.,,9STAR syndrome is an ultra-rare X-linked dominant disorder originally coined by Unger et al. as an acronym describing the characteristic phenotype of this syndrome: syndactyly, telecanthus, anogenital, and renal malformations.STAR syndrome can be associated with ocular abnormalities. Ocular manifestations include telecanthus and eyelid anomalies, in addition to peripheral anterior synechiae in the anterior segment.2This female patient was born at term  with a birth weight of 2550 g by Cesarean section for breech presentation after a pregnancy complicated by oligohydramnios. Family history was unremarkable and parents denied consanguinity. At birth, the patient was noted to have multiple congenital abnormalities, including syndactyly of the right and left 3rd, 4th, and 5th toes, telecanthus, anteriorly displaced and imperforate anus, bilateral hydronephrosis, ano-vaginal fistula, tethered spinal cord, left sensorineural hearing loss, clitoromegaly, accessory nipple, and bilateral club feet. Additional diagnostic studies revealed a ventricular septal defect and patent ductus arteriosus which closed without intervention. She was also found to have hydrocephalus but did not require a shunt placement.The patient underwent the following surgical procedures: release of tethered spinal cord at 3 months, repair of ano-vaginal fistula at 4 months, laparoscopic Ladd's procedure with appendectomy for intestinal malrotation noted at 6 months, and ileostomy and multiple surgical revisions for neurogenic bladder and bowel at 4 years of age.An ophthalmological examination was performed at an outside pediatric ophthalmology service at age 7 months; that exam noted mild telecanthus and pigment mottling in the macula of both eyes. She was examined again at 1 year and 2 years of age and was observed to have similar bilateral pigmentary changes in the macula.The patient then presented at our institution at 5 years of age for ophthalmological examination. At this visit, visual acuity was 20/200 in the right eye and 20/160 in the left eye with correction. Cycloplegic refraction was −3.50 + 4.50 × 095 in the right eye and −3.25 + 3.75 × 080 in the left eye. External examination revealed mild telecanthus, and anterior segment examination was normal in both eyes. Intraocular pressure was 14 mmHg in the right eye and 15 mmHg in the left eye.On posterior segment examination, the optic nerves were normal without evidence of optic nerve hypoplasia. Retinal examination was remarkable for yellow drusen-like deposits in the macula  and periFAM58A, which established the diagnosis of STAR syndrome. Besides FAM58A, none of the other genes were associated with any previously reported ophthalmic manifestations. The parents also underwent genetic testing and were not found to have the same mutation.Microarray analysis revealed a likely pathogenic 150 kilobase deletion of chromosome Xq28 x1) containing 6 genes including 3A phenotypic spectrum of ophthalmic manifestations has been described in patients with STAR syndrome. Telecanthus was defined by Unger et al. as a cardinal characteristic feature of this syndrome.In the posterior segment, reported optic nerve anomalies of STAR syndrome include peripapillary atrophy and optic nerve hypoplasia.In our patient, the primary ocular manifestation was retinal in the form of macular yellow pigmentary changes and peripheral, radial RPE changes. Interestingly in our patient, the macular findings appeared similar to those depicted in the fundus photos in Orge's case series of a mother-daughter pair, which the authors described as “soft macular drusen”.FAM58A gene encoding Cyclin M (CycM) lead to a deficiency in the formation of the CDK10/CycM protein kinase, which plays a key role in the regulation of ciliogenesis.FAM58A gene causes haploinsufficiency, leading to intrauterine mortality in males. A primary ciliopathy may be the mechanism by which STAR syndrome causes retinal, renal, anogenital, and digital anomalies. Guen and colleagues discovered abnormal, elongated cilia and dilated renal tubules in a kidney biopsy of a STAR syndrome patient, suggesting the presence of a ciliogenesis defect.cyclin D1 knockout mice. A limitation of our report includes the possibility that the retinal findings were related to a contiguous gene syndrome, as the deletion of Xq28 x1) contained regions neighboring FAM58A.The underlying pathogenesis in this rare genetic disorder is currently not well-understood but may be related to regulation of the cell cycle.4This case report presents the first documented multimodal imaging findings of retinal manifestations in STAR syndrome. OCT imaging revealed foveal hypoplasia as well as outer retinal atrophy and ellipsoid zone disruption suggestive of a retinal degeneration. Fundus autofluorescence imaging can highlight a characteristic, radial pattern of hyperautofluorescence as seen in other X-linked inherited retinal disorders. Questions remain as to whether the retinal changes are progressive, and further studies and long-term ophthalmic follow-up of patients with STAR syndrome are warranted.The patient's legal guardian consented to publication of the case in writing.All authors attest that they meet the current ICMJE criteria for Authorship.No funding was received for this work.We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.We further confirm that any aspect of the work covered in this manuscript that has involved human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.IRB approval was obtained (required for studies and series of 3 or more cases).Written consent to publish potentially identifying information, such as details or the case and photographs, was obtained from the patient(s) or their legal guardian(s).No conflict of interest exists.


Four Chlorella vulgaris (C. vulgaris) biomasses and one mixture of C. vulgaris and two macroalgae biomasses (Ulva rigida and Fucus vesiculosus) were used. The C. vulgaris strain was subjected to random mutagenesis and different culture conditions , resulting in different pigmentations and biochemical compositions. Couscous samples were characterized in terms of nutritional properties, oscillatory rheology properties and digestibility. All biomasses provided a significant supplementation of nutrients and excellent acceptance. The enrichment resulted in lower firmness, higher viscoelastic functions (G′ and G″) and a significant improvement in the cooking quality. Major differences between couscous samples with different microalgae were observed in protein and mineral contents, fully meeting Regulation (EC) No. 1924/2006 requirements for health claims made on foodstuffs. The amount of digested proteins was also higher in algae-containing samples. The fatty acid profile of the enriched couscous varied in a biomass-specific way, with a marked increase in linolenic acid (18:3 ω3) and a decrease in the ω6/ω3 ratio. Sensory analysis revealed that microalgae-containing products could compete with conventional goods with an added advantage, that is, having an ameliorated nutritional value using algae as a “trendy” and sustainable ingredient.The aim of this work was to develop functional couscous in a traditional Tunisian manner (hand rolling), enriched in algae biomass (6%  The link between diet and health is currently growing very steeply. People are becoming used to the consumption of sophisticated and innovative products. This is caused by many factors such as increased health care costs and population aging, thus increasing consumers’ interest in attaining a state of well-being through diet. The new way of conceiving foods originated the so-called functional products and induced a higher consumer demand for these types of goods. Currently, greater focus is being placed on sustainable “green” sources of food that produce compounds with efficient biological activity, which could be used as functional ingredients or dietary supplements. These biomolecules include polyunsaturated fatty acids (PUFAs), proteins, vitamins and minerals ,2.Arthrospira (a cyanobacteria commonly known as Spirulina) as food [Algal biomass and products thereof figure at the top of the list in the food market as sustainable nutritional bio-ingredients and are promoted as “superfoods” which could be used in the formulation of “trendy” goods . Consump as food . In rece as food ,6,7, pas as food , milksha as food , and som as food . This ma as food . FurtherAlgae biomasses comprise a wide spectrum of bioactive compounds which vary mainly depending on the strain and the culture conditions, such as the physiological status and growth phase . The difThe organoleptic characteristics of algae biomass are strongly linked to the consumers’ acceptance since color is the first parameter taken into consideration by consumers and can be decisive for the inclusion of the food in their diet . TherefoChlorella vulgaris (C. vulgaris) biomass with different pigmentations. A mixture of microalgae (C. vulgaris) and seaweeds (Fucus vesiculosus and Ulva rigida) was also used. It was predetermined to use an elevated concentration of algal biomass compared to the concentrations typically present in commercial products (<1%) in order to bring higher levels of bioactive molecules that are sufficient to functionalize the product. The food matrix in which the algae biomass was incorporated was couscous, an ethnic staple food product of some of the Maghreb countries or the Berber world (Northern Africa). This cereal product is known for its simplicity in terms of the raw materials used and the cooking process, in addition to its nutritional characteristics such as its low fat content and the presence of some vitamins and minerals [The aim of the current work was to develop a novel food product with a high concentration (6%) of minerals . A few sminerals ,19; someminerals . Likewisminerals .The current paper discusses the overall quality and the acceptability of the end product. The variables evaluated include color, texture and mechanical features. In vitro gastrointestinal digestion was performed to mimic physiological conditions to evaluate the degree of nutrient bioaccessibility. A sensory analysis that included assessment of flavor, overall acceptance and purchase intention was conducted.Chlorella vulgaris AGF002 (C. vulgaris) and Algaessence® biomass were provided by Allmicroalgae Natural Products . C. vulgaris was cultivated in auto- and heterotrophic modes. Guillard’s F2 medium [® CelliGen®115; Eppendorf AG, Hamburg, Germany), using glucose as the organic carbon source with a C: N ratio of 6.7:1. Chemically random mutagenesis was induced in order to develop chlorophyll-deficient C. vulgaris mutants with different pigmentations [C. vulgaris (Organic Chlorella) and heterotrophic C. vulgaris: Smooth Chlorella (light green color), Honey Chlorella (yellow color) and White Chlorella (white color). Algaessence® is a micro- and macroalgae blend composed of Organic Chlorella, Fucus vesiculosus and Ulva rigida. Macroalgae biomasses were cultivated by ALGAplus , and the mixture was packaged and commercialized by Allmicroalgae Natural Products. Except for the mineral profile and chlorophyll content, the chemical composition of the different algae biomasses used was provided by Allmicroalgae as indicated in 2 medium  was adjuMedium semolina (100% durum wheat) from “Pâtes Warda”  was used for couscous production .wt/wt) of semolina was replaced by the different types of algae. The flour was first moistened with water (40%) in a dish, followed by circular movements to ensure an adequate homogenization. The moistened semolina then underwent the sizing and sieving steps, using three sieves with mesh openings decreasing from 1280 to 1000 µm. Sizing aims to force all the particles through the holes of the sieve, by moving and pressing them with the palm of the hand. No agglomerated couscous particles (<1000 µm) were recycled, and rolled couscous grains up to 1000 µm were dried at 30 °C/24 h . The granulometry of the dried couscous was controlled by sieving on 500 µm and 1130 µm sieves in order to eliminate fine and large particles. Three batches of 100 g of couscous dough were developed for each formulation. The dry couscous was rehydrated (40% of water) and rolling movements were applied for 10 min, in order to help the grains absorb the water sufficiently. The hydrated granules were placed in the superior section of a couscoussiere (steaming pot), while the lower section contained boiling water. The steaming lasted 30 min. The steam cooking procedure is considered the last step and was repeated twice. The steam-cooked couscous was set to cool down and then stored at −20 °C.Couscous was produced employing an optimized traditional Tunisian handmade procedure, and all the steps are summarized in The cooking quality variables of the couscous samples were determined in terms of cooking loss (CL), swelling, water absorption index (WAI) and water solubility index (WSI). The swelling was determined as described by Ounane et al. , by mixiThe color of uncooked and steam-cooked samples was instrumentally  evaluated according to CIELAB system color parameters , where L* indicates brightness , a* indicates the degree of redness or greenness  and b* indicates the degree of yellowness or blueness . The measurements were conducted under the same artificial fluorescent light using a white standard , and the same temperature (20 ± 1 °C). The measurement was replicated at least eight times.The small amplitude oscillatory shear (SAOS) behavior of the couscous dough (before sieving) was measured in a controlled-stress rheometer  with a UTC-Peltier system. The sample was placed on the bottom of a serrated parallel-plate sensor with a 35 mm diameter (PP35) and a 3 mm gap (previously optimized). After placing the rheometer in the measuring position, with a 3 mm gap between plates, the edges were coated with liquid paraffin to prevent moisture losses during tests. The linear viscoelastic region (LVER) was previously accessed for all the samples, at 6.28 rad/s (1 Hz), through a stress sweep test, and in all the samples, a constant shear stress of 20 Pa was applied to perform the frequency sweep tests (from 0.0628 to 628 rad/s). All rheology measurements were repeated at least three times. Storage moduli (elastic) G′ and loss moduli (viscous) G″ (Pa) data versus angular frequency ω (rad/s) were fitted through power equations, where α′, α″, b′ and b″ are the corresponding fitting parameters (Equations (1) and (2)) .G′ (ω) −1 crosshead speed. Before each test, the couscous grains were cooked as described above and left to cool down to 20 °C for 1 h. The steam-cooked couscous (20 g) was placed in cylindrical glass cases (4 cm diameter and 4.2 cm height) and measurements were repeated at least six times for each sample. Resistance to penetration was expressed as firmness (N) and corresponds to the maximum force value.The couscous texture was evaluated through a texturometer, TA-XTplus , in compression mode (50%) using a cylindrical aluminum probe with a diameter of 30 mm and 9 mm of penetration distance at 1 mm·s2 gas. The mineral profile was evaluated by inductively coupled plasma optical emission spectrometry  [Moisture and ash content were determined conforming to ICC methods 110/1 and 104/1, respectively. The total fat content was measured by acidic hydrolysis, followed by centrifugation and extraction with n-hexane. The crude fat residue was determined gravimetrically, after rotary evaporation and N series) . The cruw/w, dry basis.Concerning pigment determination, aliquots of algal extracts were prepared at 1 mg/mL (in ethanol), absorbances were measured at 470, 648 and 664 nm and then the chlorophyll content was calculated using the Lichtenthaler equations . Fatty awt/wt) with simulated salivary fluid (SSF) containing amylase (300 U/mL) for 2 min at pH 7. The mixture was then diluted with simulated gastric fluid (SGF) containing gastric enzyme (Pepsine 2000 U/mL) and incubated for 2 h (pH 3). Subsequently, simulated intestinal fluid (SIF) containing pancreatin (100 U/mL) and bile salts (20 mM) was added and incubated for a further 2 h (Ph 7). The whole digestion protocol was performed at 37 °C, under constant gentle mixing on a rotating wheel. Finally, the intestinal phase was stopped using the protease inhibitor 4-(2 aminoethyl) benzensulfonylfluorid . A reagent blank was also prepared. All samples were finally centrifuged , and the undigested residues were collected and dried at 80 °C for 6 h, and then at 45 °C, until constant weight.The cooked couscous samples’ in vitro digestibility was evaluated following the INFOGEST protocol . The enzThe dry matter and crude protein in vitro digestibility (%) of all the couscous products was calculated from the difference between the initial biomass and the undigested biomass, divided by the initial biomass and multiplied by 100. Analyses were repeated in triplicate.Chlorella commercial biomasses at a 6% (wt/wt) incorporation level. The test was conducted in a standardized sensory analysis room as previously described by Khemiri et al. [Sensory analysis assay was performed for couscous enriched with Honey, Smooth and Organic i et al. . Samplesp < 0.05) of treatments were detected in ANOVA, multiple comparisons of means were conducted using Tukey’s approach. Principle component analysis (PCA)  was carried out to achieve a better understanding of the relationship between the different sensory attributes. All results are reported as average ± standard deviation (SD).Statistical analysis was performed using Origin Pro 8.0 software , through analysis of variance (one-way ANOVA). When significant effects  lower values  is visually very attractive, presenting yellow or different shades of green  within the same color parameter. Color losses resulting from the couscous cooking process are related to pigment oxidation, besides a certain degree of leaching [The presence of algae biomass in the formulation of couscous led to variations in color compared to the control couscous , as expeof green B. The coof green . The higleaching ,29. The Chlorella with the highest protein contents  increases [The results from the small amplitude oscillatory shear (SAOS) measurements of the uncooked couscous are expressed in terms of storage (G′) and loss (G″) moduli . All thecontents . It appes et al.  studied ncreases . It is wp < 0.05) decrease in the cooked couscous firmness (9–14 N) in comparison with the control sample (16 N). The difference in firmness mainly arose from the difference in the gluten fraction. An increase in the gluten content results in a structural reinforcement and vice versa. In the current investigation, decreasing the gluten amount by replacing semolina with algae biomass resulted in structure weakening. The decrease in the firmness was significantly noticeable in couscous with Smooth and Honey Chlorella with the lowest protein content  between the Control, White and Organic couscous firmness values. Additionally, the softness obtained in the Algaessence couscous could be due to the high fiber content in the Algaessence biomass . Fiber dwt/wt) of semolina with algae biomass. As predicted, the chosen ratio permitted product functionality. Couscous samples showed varying chemical compositions depending on the types of algae used  than the control sample , with advantages in terms of nutritional performance. As far as the mineral content is concerned, a significant improvement in major and trace minerals was determined  of steam-cooked couscous is presented in Chlorella and Algaessence® biomass to couscous may be an appealing way to increase the daily intake of functional nutrients, such as minerals and polyunsaturated fatty acids. This supplementation would promote and prevent different types of disorders such as gastric ulcers, constipation, anemia, hypertension, diabetes, infant malnutrition and neurosis [The addition of neurosis ,37.Arthrospira platensis to gluten-free pasta corresponded to a significant decrease in digestibility. Although the dry matter IVD was similar for all couscous samples, differences emerged in protein digestibility. Couscous supplemented with Honey and Organic Chlorella presented a significant increase in IVD, while couscous with Algaessence® biomass was similar to the control sample. The in vitro digestibility of microalgae-based foods may vary according to the species, cell wall structure and nutritional composition [Digestibility is an important factor in determining the level of nutritive factors. Thus, if algae are to be used as functional ingredients, it is crucial to know their impact on digestibility. Only few papers have studied the in vitro digestibility (IVD) of algae-based foods ,38. To oChlorella biomasses. The selection of the samples was based on protein digestibility results, and also on gaining a better assessment of the impact of pigment colors in the sensory assays. Khemiri at al. [Sensory analysis of enriched couscous highly contributes to its potential future commercialization, since it provides a perspective of the potential product’s acceptability in the market. Couscous with microalgae biomass presented an attractive and innovative appearance, as seen in i at al. ,39 and Ni at al.  recentlyPCA was carried out to evaluate the efficiency of the nine-point hedonic scale on the classification of the different sensory attributes of the tested samples and to better understand the relationship between the attributes within the same sample. The sensory attributes of the Honey and Smooth couscous samples are positioned on the right side of the score plot, while the Organic sample attributes are grouped on the left. The loading plot (biplot) shows that the first principal component, which mainly separates the Honey and Smooth samples from the Organic couscous, is dominated by the highest “like” levels on the hedonic scale (like very much and like extremely) on one side and the highest “dislike” levels  on the other side. This means that the first component of the data, accounting for 36% of the total variability, was effective in separating the samples from each other based on the extreme degree of preference. The texture and color of the Organic sample are better described by the second principal component, which explains 21.74% of the total variability. Additionally, sensory attributes of the samples with the same degree of preference are clustered together. Some sensory attributes, more than others, contribute to the overall acceptability of the product. In the case of the Honey and Smooth couscous, the flavor and the color were the most influential attributes for global appreciation since they have close scores along PC1. As far as the Organic couscous is concerned, the global appreciation of the sample is related to the flavor and odor. The flavor and the odor of the Organic couscous were least appreciated and were highly correlated on the basis of PC1. The results of consumers’ intention to buy the newly formulated couscous are presented in w/w) to semolina flour. These products boast healthy properties imparted by the presence of functional biomolecules while maintaining the typical versatility of traditional couscous. Changes observed in the biochemical composition of the used biomass, due to different strain-specific characteristics and different culture conditions, led to couscous with different appearances and nutrient contents. The contents of protein , iron and magnesium (at least twice the RDV) were compatible with the provisions of Regulation (EC) No. 1924/2006, fulfilling the nutritional and health claims related to biocompounds. Additionally, the enrichment of couscous with algae permitted a higher protein in vitro digestibility and a clear improvement in the omega-ω6 to omega-ω3 fatty acid ratio, although it also increased the proportion of saturated fatty acids. Moreover, the innovative products exhibited acceptable cooking and sensory properties. Therefore, their position in the market of functional products is conceivably lucrative for food industries and algae biomass producers, which would, in turn, develop and commercialize functional couscous enriched in algae biomass.Innovative couscous products were successfully produced by adding algae biomass (6%


Collectively, these mechanisms act to protect against stress, injury, and the detrimental effects of chemicals on mitochondria. In this study, we examined the effects of cocaine on cardiac mitochondrial dynamics, biogenesis, and UPRmt in vivo. Rats administered cocaine via the tail vein at a dose of 20 mg/kg/day for 7 days showed no structural changes in the myocardium, but electron microscopy revealed a significant increase in the number of cardiac mitochondria. Correspondingly, the expressions of the mitochondrial fission gene and mitochondrial biogenesis were increased after cocaine administration. Significant increase in the expression and nuclear translocation of activating transcription factor 5, the major active regulator of UPRmt, were observed after cocaine administration. Accordingly, our findings show that before any structural changes are observable in the myocardium, cocaine alters mitochondrial dynamics, elevates mitochondrial biogenesis, and induces the activation of UPRmt. These alterations might reflect cardiac mitochondrial compensation to protect against the cardiotoxicity of cocaine.Abuse of the potent psychostimulant cocaine is widely established to have cardiovascular consequences. The cardiotoxicity of cocaine is mainly associated with oxidative stress and mitochondrial dysfunction. Mitochondrial dynamics and biogenesis, as well as the mitochondrial unfolded protein response (UPR Sufficient evidence shows that the use of cocaine is related to a series of dangerous cardiovascular consequences, such as coronary artery spasm2, dysrhythmia4, hypertension5, left ventricular hypertrophy6, and myocardial infarction8. In addition to the anesthetic effect of cocaine by blocking sodium channels, the cocaine-induced cardiovascular disorders are mainly due to its potent sympathomimetic effect, which is achieved by the blockage of catecholamine reuptake at neuronal presynaptic terminals10. This allows cocaine not only to escalate the myocardial oxygen demand, but also, simultaneously, to decrease the myocardial oxygen flow12. In general, oxidative stress and mitochondrial dysfunction in cardiomyocytes are believed to play pivotal roles in cocaine-induced cardiotoxicity17.Cocaine (benzoylmethylecgonine), a strongly addictive psychostimulant, is the second most commonly used recreational drug in the world20. Moreover, mitochondria are implicated in the maintenance of intracellular Ca2+ homeostasis, the regulation of cell death and survival, and are the primary origin of the reactive oxygen species (ROS) that trigger oxidative stress when overproduced23. Studies have found that mitochondria play a key role in cardiovascular pathogenesis, such as dysrhythmia and heart failure25. Thence, the homeostasis and normal function of mitochondria are crucial for the ideal operation of the heart, and these are sustained by mitochondrial dynamics and biogenesis26.Cardiac mitochondria, which occupy 20–40% of the cardiac cellular volume, are indispensable for producing the copious amounts of ATP required to sustain the unremitting contraction of the myocardium28. Briefly, optic atrophy 1 (OPA1) and mitofusin (MFN) 1 and 2 facilitate mitochondrial fusion29. On the other hand, dynamin-related protein 1 (Drp1), which plays a leading role in mitochondrial fission, is phosphorylated and recruited to the fission point. Phospho-Drp1 (p-Drp1) then promotes the formation of a membrane constriction ring by reactions with receptors and Drp1 oligomerization, which then leads to the completion of mitochondrial fission31. Mitochondrial biogenesis, on the other hand, is driven by peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α)32. Effectors of PGC-1, such as PPARα, as well as transcription factors such as nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) also participate in the regulation of mitochondrial biogenesis33. Mitochondrial dynamics and biogenesis form a delicate coping mechanism to maintain the optimal energic output of mitochondria in response to different environments, including increased oxidative stress and toxic conditions35. They are closely associated with the homeostasis of the cardiovascular system and an altered balance of mitochondrial dynamics can lead to pathological cardiac remodeling and cardiac pathogenesis38. Accordingly, the status of mitochondrial dynamics and biogenesis is fascinating when against the cardiotoxicity of cocaine. Recently, the protective role of the mitochondrial unfolded protein response (UPRmt) in cardiac pathophysiology has drawn increasing attention. During the accumulation of mitochondrial stress, activated UPRmt promotes the recovery of the oxidative phosphorylation machinery39, restores proteostasis40, and detoxifies excess ROS41, accomplished by the translocation of its major regulator, activating transcription factor 5 (ATF5), and the activation of mitochondrial resident chaperones and proteases43. It remains unrevealed whether UPRmt participates in resistance to the cardiotoxicity of cocaine.Mitochondrial dynamics, which refers to the continuous fusion-fission circulation of mitochondria, is regulated by a series of dynamin-related GTPasesmt was shown to be activated, although only a panel of its downstream effector gene expressions were indeed upregulated. We believe that this is the first in vivo study showing the altered mitochondrial dynamics and biogenesis, as well as the involvement of PPARα and UPRmt, in cardiomyocytes after cocaine administration.Here, we investigate whether cocaine affects cardiac mitochondrial dynamics and biogenesis in vivo. After rats were administered 20 mg/kg/day cocaine via the tail vein for 7 days, we found strengthened cardiac mitochondrial fission and biogenesis, as well as the recruitment of PPARα, while the expressions of mitochondrial fusion factors were decreased. In addition, UPRAll methods were carried out in accordance with relevant guidelines and regulations.mt factors and PPARα, and to obtain samples for transmission electron microscopy, a control group (n = 4) and a high dose cocaine group (n = 7) were established under the same condition as the first-round experiments. Findings of the two rounds of experiments are shown collectively to provide a comprehensive demonstration of the consequences of cocaine cardiotoxicity.All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University. All animal work was performed in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and regulations. Male Sprague Dawley rats (8 weeks old) were randomly divided into 3 groups  and administered saline (control group) or cocaine [low (5 mg/kg)/ high (20 mg/kg) dose cocaine group] in the first-round experiments. In second-round experiments to further examine expressions of oxidative phosphorylation system proteins, complex I activity, the protein expressions of UPR45. Cocaine hydrochloride  was dissolved in saline and administered intravenously via the tail vein  for 7 days. Control rats received the same volume of saline injected via the tail vein on the same schedule. The rats were sacrificed by an overdose of sodium pentobarbital  24 h after the last administration. The hearts were immediately harvested and placed in phosphate-buffered saline solution . Subsequently, the left ventricles were evenly divided for further histological and biochemical analyses.Dose conversions from humans to rats were made to establish the conditions of 5 and 20 mg/kg/day to represent typical serum concentrations for recreational cocaine abusers and beneath the concentrations for acute cocaine poisoning overdoseThe left ventricular specimens for microscopy were fixed in 4% paraformaldehyde, embedded in paraffin, and 2.5 µm-thick sections were prepared. Hematoxylin–eosin (H&E) and Elastica-Masson-Goldner (EMG) staining were performed for histological analysis. Immunohistochemical staining of PPARα and ATF5 were conducted. Briefly, sections were deparaffinized in xylene, rehydrated with a series of graded ethanol, and heated in 10 mM citrate buffer (pH 6.0) for antigen retrieval. 3% hydrogen peroxide was applied afterward to quench the activity of endogenous peroxidase. The sections were then blocked with 0.5% bovine serum albumin serum in PBS. Subsequently, the sections were incubated with rabbit anti-ATF5 monoclonal antibody  or mouse anti-PPARα monoclonal antibody  overnight at 4 °C, followed by visualization of antigens by use of Histofine simple stain MAX-PO (Multi)  using diaminobenzidine (Nichirei Biosciences Inc.) as a substrate. Sections incubated in PBS in place of the primary antibodies were used as controls for immunostaining procedures.The left ventricle specimens for mitochondrial complex I enzyme activity analysis were immediately frozen to − 80 °C. Mitochondria were extracted from the specimens using a Mitochondria Isolation Kit . The protein concentrations of the extracts were determined by Bicinchoninic Acid protein assay and adjusted to 100 µg/mL with PBS. Subsequently, a Complex I Enzyme Activity Assay Kit  was used to determine the activity of the electron transport chain (ETC) following the manufacturer’s protocol. The absorbance at 450 nm was measured using a microplate reader .3VO4, and protease inhibitor cocktail ), and the lysates were subjected to SDS–polyacrylamide gel electrophoresis. Immunoblotting was then performed with voltage-dependent anion channel 2 (VDAC2) antibody , the translocase of the outer mitochondrial membrane 20 (TOM20) antibody, , total OXPHOS Rodent WB antibody cocktail , anti-PGC-1 polyclonal antibody , anti-NRF1 antibody , anti- TFAM antibody , anti-PPARα monoclonal antibody , anti-ATF5 rabbit monoclonal antibody (Abcam), anti-CHOP mouse monoclonal antibody , and anti-actin antibody . Antigens were visualized by peroxidase-bonded anti-Mouse or rabbit IgG secondary antibodies  and enhanced chemiluminescence reagents . The band densities were quantified by CS analyzer 4 image analyzing software .The left ventricle specimens for immunoblotting were lysed in STE buffer  and fixed with 4.5% paraformaldehyde and 2.5% glutaraldehyde in PB. After incubated in 1% osmium tetroxide for 2 h, the fixed specimens were dehydrated in a series of graded ethanol and embedded in Epon epoxy resin. Ultrathin sections of the embedded specimens were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope  and AMT Advantage-HS CCD camera . The number of mitochondria per field  was counted from 10 electron micrographs in each group. Randomly selected cross-sectional areas of mitochondria (n = 100 for each group) in cross-sectional views of cardiac myofibrils were counted using ImageJ software (ver1.53).46. The primers used are listed in Supplementary Table Real-time reverse transcriptase-mediated PCR analysis was conducted as described previouslyp < 0.05.Student’s t-test and Dunnett’s test were used to assess statistical significance throughout this study. Statistical differences were considered significant at Cocaine has been reported to induce cardiovascular disorders in animal models via intraperitoneal administration. To assess the effect of cocaine on the myocardium in our model using tail vein administration, H&E and EMG staining of left ventricular specimens from the control group and cocaine group rats were conducted Fig. . The myoNext, we determined the effect of cocaine on cardiac mitochondrial status and function. Immunoblotting of mitochondria oxidative phosphorylation subunit proteins showed an increase in the complex I protein. Apart from complex I and IV proteins, the expressions of complex II, complex III, complex V, VDAC2, and TOM20 showed no significant changes after cocaine administration  via tail vein in our experiments. This might be owing to the relatively short experimental period of our study. Additionally, although there are no comparative studies on toxicity of cocaine administered via different routes, one report has suggested a lower toxicity of gold nanoparticles administered via the tail vein as compared to intraperitoneal injection52. As a result, it is also possible that our negative histological findings of structural changes in myocardium were due to the influence of the different administration route of cocaine as compared with previous researches. We further investigated the status of cardiac mitochondria after cocaine administration because mitochondrial dysfunction is one of the hallmarks of cocaine cardiotoxicity. These examinations revealed normally reserved mitochondrial status and function. The function of complex I, the first and largest enzyme of ETC, and most of its core subunits, which are mitochondrially encoded proteins53, were elevated rather than impaired after 7 days of cocaine administration via the tail vein. It has been repeatedly reported that cocaine-induced oxidative stress increases the production of ROS, damages the ETC, suppresses the generation of ATP54, as well as compromises the antioxidative system54, and all of these eventually results in cardiac mitochondrial dysfunction55. Animal models of cocaine abuse have also revealed an extensively decreased expression of mitochondrial genes and proteins57. The rare but meaningful findings in our study showed a still normally maintained mitochondrial status after cocaine administration, which suggests that a mitochondria-targeted self-compensation mechanism against cocaine cardiotoxicity was activated prior to the occurrence of any structural changes in myocardium.Abundant evidence implicates cocaine abuse in cardiovascular disease. The clinical short-term consequences of cocaine intoxication include acute myocardial infarctionmt help to preserve mitochondrial homeostasis in cardiomyocytes and act protectively against detrimental effects. In particular, mitochondrial dynamics and biogenesis take part in determining mitochondrial quality and abundance, thereby maintaining adequate myocardial function61. Elevated mitochondrial fission and biogenesis meet the increased energy demand of cardiac myocytes under stress or ischemic injury or the harmful effects of chemicals62. We discovered that it is the same circumstance in cardiac mitochondria after cocaine administration. In this study, we found the activations of mitochondrial fission and biogenesis, together with downregulated mitochondrial fusion gene expression. These effects may act together to profoundly increase the number of mitochondria, as we observed by electron microscopy, so as to maintain normal cardiac function under the detrimental effects of cocaine. Mitochondrial Fis1 recruitment has been demonstrated as an early event in the cardiac pathological process64, and the elevated fission machinery ultimately results in cardiomyopathy26. It is consistent with our view of the mitochondrial compensatory stage ahead of any pathological changes. In addition, during pathological cardiac remodeling, TFAM protects the mitochondrial DNA from mutation, and its overexpression is beneficial to the recovery of cardiac function65. It is likely that this protective effect of TFAM might also participate in the specific upregulation of mitochondrial DNA and preserved function of complex I seen in this study. Meanwhile, as the effector of PGC-1, PPARα also participates in mitochondrial biogenesis and homeostasis because of its central role in fatty acid oxidation and other lipid metabolism events68. However, the involvement of PPARα in the cardiotoxicity of cocaine has not been illustrated. Our immunohistochemical observation indicates the recruitment of PPARα in the myocardium after cocaine administration. This might be related to our observation of elevated mitochondrial biogenesis in cocaine group rats and implies a potential role of PPARα in resistance to the cardiotoxicity of cocaine. UPRmt is another potential protective mechanism to explain the unimpaired mitochondrial function we observed. UPRmt activation has been shown to be important for the normal function of cardiac myocytes43. Among numerous involved factors, ATF5 should be involved in the UPRmt cardioprotective effects against stress, cardiac dysfunction, and pathological remodeling, because its activation and nuclear translocation trigger the downstream responses of UPRmt70. CHOP and Hsp60, on the other hand, play key roles in maintaining mitochondrial homeostasis in UPRmt71. Our examination of UPRmt revealed an interesting phenomenon that instead of all UPRmt factors being collectively upregulated after cocaine administration, the expressions of several UPRmt factors decreased significantly. It has been reported that Lon regulates mitochondrial transcription by selectively degrading TFAM72, and the absence of CLPP reduces mitochondrial cardiomyopathy73. These findings suggest that the downregulation of LONP1 and CLPP that we observed might also play a protective role against cocaine cardiotoxicity. Nevertheless, further research will be required in order to fully understand the paradoxical expression of UPRmt factors that we observed. So far, our findings indicate that UPRmt participates in cardiac mitochondrial protection against the cardiotoxicity of cocaine, even if not complete and collectively activated at an early stage.As stated above, mechanisms such as mitochondrial dynamics, biogenesis, and UPRmt in the myocardium. The limit of this study is the insufficient data of cardiac and mitochondrial functional changes, as well as stress accumulation after cocaine administration, which necessitates more functional assessments of heart and mitochondria in following studies, such as echocardiography, respiration profiles, and ATP production rates. It is worth noticing that the differences might present among individual models, but our findings should be beneficial for an advanced understanding of the cardiotoxicity of cocaine, especially the early events following cocaine exposure.In conclusion, we demonstrated that 7-day 20 mg/kg/day cocaine administration to rats via the tail vein results in elevated cardiac mitochondrial fission and biogenesis, the recruitment of PPARα, and the activation of mitochondrial protecting UPRSupplementary Information.


The application potential of functional ingredients for the meat industry is often assessed through different measuring tools, thereby making comparisons difficult. The aim of this study was to create valuable information about the performance of functional ingredients based upon standardized and comparable data gathered through a newly developed screening tool. Therefore, 25 ingredients, selected from different techno-functional classes, were characterized at 2 different dosages by means of the screening methodology. The tool itself consisted of a lean meat model and fatty liver-based system, representative of the finely minced and/or emulsified charcuterie market. A total of 23 different parameters were measured through both model systems, providing information concerning water and fat binding capacity, emulsification, and texture and structure formation. Through cluster analysis, the ingredients were assigned to groups, each with their own specific properties. The screening tool provided good descriptive and distinctive power concerning ingredient functionalities and offers the industry a clear overview of their application characteristics. Processed meat products often contain so-called techno-functional ingredients that are added to improve product quality, reduce formulation costs, satisfy consumer needs , and to keep up with current market trends, such as clean label, fat reduction, and salt reduction. Balestra and Petracci  indicateFirstly, ingredient manufactures often rely on water-based model systems to describe the characteristics of their products. Yet, it is well-known that ingredients often behave differently in a watery environment compared to a meat matrix. The importance of a meat matrix to measure ingredient functionality was described by Balestra and Petracci . A food Many authors ,11,12,13Furthermore, a comprehensive literature study revealed that functional ingredients often contribute to similar quality characteristics  in meat products, despite their difference in nature, origin, or product class. Therefore, classification of ingredients (at specific dosages) based upon similar behavior rather than product class could be more beneficial or practical for the meat industry. As mentioned before, a standardized and uniform measuring tool is hereby of great value. Therefore, the goal of this study is to gather key information concerning the characteristics of 25 techno-functional ingredients, each applied at two dosages in a meat matrix based upon a newly developed screening tool. The obtained standardized and scientifically collected data are statistically processed to cluster ingredient x dosage combinations in groups exerting similar performances. This will lead to better insights and understanding of their functionalities, and thus, better estimation of the use of certain ingredient x dosage combinations in specific meat applications.A lean meat model (LMM) and fatty liver-based model system (FLM) were developed and validated to serve as a standardized screening tool for the measurement of functional ingredient characteristics. They represent the wide range of both lean (cooked ham) and fatty heated meat products available in the market. Altogether, 23 different parameters (see following sections) were measured through the model systems.Longissimus dorsi muscle for LMM; liver and back fat for FLM) and water.In both LMM and FLM, a total of 25 ingredients were tested. The choice of ingredients was based upon a short inquiry of the Flemish meat industry and was mainly directed by their common practice. Ingredients were derived from different functional classes and fixed low (LD) and high dosage (HD) levels were defined for each class, based on ingredient product sheets and literature . The LD To match the scientific goals to the available experimental material and time, the experiments were optimized in such a way that replication was maximized for each treatment , and this was the case for both model systems, taking into account the different ingredient classes that were considered . In addiL. dorsi muscle, pork liver, and pork back fat) were purchased from a local supplier. The entire experiment was performed using a single homogeneous batch of each respective raw material. Back fat was cut into small cubes, and L. dorsi muscle and liver were ground through an 8 mm plate. All raw materials were each mixed thoroughly to obtain a large uniform batch of each raw material, packed as smaller portions into plastic bags and frozen (−18 °C) until use. The day before preparation of the specific model systems, raw materials were thawed overnight in a refrigerator at 4 °C. This approach allowed us to study only the impact of the ingredients without interference of potential variability in raw material characteristics.LMM and FLM model systems were prepared in the pilot plant of the research group ‘Technology and Quality of Animal Products’ . Raw materials (pork L. dorsi muscle (40 g/100 g) and ice-water (60 g/100 g), in which nitrite curing salt (1.6 g/100 g) and tetrasodium pyrophosphate (0.2 g/100 g) were dissolved in advance. Auxiliary ingredients were calculated relative to the total mass of meat raw materials (L. dorsi muscle) and water. The lean meat and brine were shortly mixed (10 s) at 2000 RPM before adding the ingredient under investigation , pork back fat (50 g/100 g), and broth obtained by scalding the back fat (35 g/100 g). It was processed by first separately prechopping the raw liver for 8 min at 3500 RPM in a Stephan UM12 vertical cutter-mixer . The liver batter was stored at 4 °C until further processing. The back fat was scalded in boiling water during 20 min. Afterwards, the scalded fat together with the broth were added to the bowl cutter and chopped at 3500 RPM during 5 min. Temperature of the obtained fat emulsion reached 50–51 °C at the end of chopping. The liver batter, nitrite curing salt (1.6 g/100 g), and ingredient under investigation  were addBoth recipes of the described model systems have a quite critical composition, meaning that they contain substantially more water and/or fat than regular meat products. These recipes were developed to induce poor product quality, allowing us to measure the effect of the different ingredients and compare them more easily.Rheological measurements of both raw LMM and FLM batters were executed using an AR2000ex stress-controlled rheometer  equipped with a 40-mm parallel plate. The upper and lower plate were crosshatched to prevent slippage of the samples. The gap between the plates was set at 1000 μm. The AR2000ex was equipped with a Peltier temperature control system and upper heated plate (TA Instruments) to control the sample temperatures precisely.G′, measurement of elastic characteristics and G″, measurement of viscous characteristics respectively) were directly obtained from the Rheology Advantage Data Analysis, v. 5.7.0 software . The LVR represents the stress range within which both G′ and G″ are independent of the imposed stress amplitude and is determined according to Glorieux et al. [LVR-LMM, G′-LMM) and FLM  are expressed logarithmically and used in the cluster analysis . The ‘storage’ and ‘loss’ modulus  meat products. The following profile was applied: (1) heating from 14 °C and 40 °C for LMM and FLM, respectively, to 76 °C at a constant heating rate of 2 °C/min; (2) isothermal time sweep at 76 °C for 3 min; (3) cooling from 76 °C to 14 °C at a constant cooling rate of 2 °C/min; (4) isothermal time sweep at 14 °C for 10 min (only for FLM). The entire process is illustrated in g by means of a Universal 320 R centrifuge  at 25 °C for 3 min of a preweighed amount of raw batter. The percentage of total expressible fluid (TEF-FLM) was expressed as follows:Emulsion stability of FLM was determined immediately after batter preparation according to Glorieux, Goemaere, Steen and Fraeye  with sliIn addition, the relative amount of water next to the fat in the drip was determined. Therefore, drip loss after centrifugation was collected and weighed before and after drying in a Typ U 40 oven  for 24 h. The relative amount of water in the drip loss was expressed as follows:TEF-FLM and 2O in drip-FLMRelative amount of H were determined in triplicate for each experimental run and used as parameters in the cluster analysis.Drip loss cold-LMM). The determination was executed in duplo for each experimental run by centrifuging 30 g of meat batter at 9526× g (9000 rpm) for 5 min at 25 °C, after which the separated fluid was weighed. Drip loss cold-LMM was expressed as follows:The drip loss of the raw batter of LMM was measured immediately after batter preparation (Drip loss hot-LMM) is similar to the cold procedure, albeit that before centrifugation at 470× g (2000 rpm) for 1 min at 25 °C the samples were heated at 75 °C for 30 min in a KK 2800 smoke-air cooker . Drip loss hot-LMM was determined in triplicate for each experimental run and expressed as follows:Drip loss cold-LMM and Drip loss hot-LMM were used as parameters in the cluster analysis.Determination of the drip loss of the batter of LMM upon heating (CL-LMM) was determined in duplo for each experimental run according to Perez-Santaescolastica et al. [Cooking loss of LMM  in plastic trays. The trays were stored upright under an angle of 45° in a cool cell at 3 °C. After 3 weeks of storage, the drip loss was collected and used as a measurement for the syneresis of the sample. Syneresis-LMM was determined in duplo for each experimental run and expressed as follows:LMM was sliced and MAP packed  and expressed as the maximum force (N) to penetrate the sample, as described in Perez-Santaescolastica, Goemaere, Hanskens, Lorenzo and Fraeye . A totalTo cluster all ingredients with the given concentration (high/low), a hierarchical cluster analysis was performed based on the 23 above-described quality parameters. Mean values of each parameter per ingredient x dosage combination were expressed relative to their corresponding average blank value by dividing both and used for further statistical processing. To define the clusters, Ward’s minimum variance method was used . DecisioAll statistical analyses were performed in JMP Pro 14 .The green cluster includes the blank (reference) to which no ingredients were added. Hence, ingredient x dosage combinations in the green cluster represent samples which have limited impact on the parameters measured compared to the blank sample. Of the five clusters identified, the green cluster groups the highest amount of ingredients, mainly ingredients at LD. The LD of each ingredient class is established, as mentioned before, based upon literature and available product sheets and corresponds approximately to the minimum dosage needed to improve product quality to some extent. As a consequence, the impact of LD ingredients on parameters measured is rather small compared to the blank.Closest to the blank sample are mainly collagen-derived proteins and plant proteins at LD. The contribution of gelatins to water binding in heated meat products is rather limited mainly due to their lack of gelling capacity at high temperature. During heating, gelatin solubilizes in the watery phase  and can In general, plant proteins have less pronounced functional properties compared to that of animal proteins in meat products. Moreover, an effective replacement of animal proteins requires certain technological innovations. New extraction and drying technologies are proposed to ameliorate functional characteristics of plant proteins such as pea protein ,18. AddiG′ heat 76 °C-FLM). As a consequence, an inferior stabilization of fat, expressed as a high increase of TEF-FLM, occurred. In case of emulsifiers, these phenomena can be explained through the different theories of fat stabilization in meat batters. The first theory states that stabilization of fat results from the formation of an interfacial protein film (IPF) around the fat globule, while the second one claims that fat globules are physically entrapped within the protein matrix [Somewhat further away from the blank sample are starches and emulsifiers. The appearance of pea fiber in that group can be easily explained due to the relatively high amount of starch in the composition of the ingredient, according to the manufacturer (not shown). All starches screened in the experiment are  native starches. In general, the functionality of native starches is rather restricted compared to chemically modified starches, which could explain their occurrence in the green cluster. Nowadays more and more starch producers have found innovative solutions to create ‘native functional’ starches by using physical processes with comparable properties to chemically modified ones ,9. The pn matrix . Meat emn matrix . During n matrix . The negn matrix  observedn matrix  also notMany ingredients in the red cluster are only included at HD, while their LD counterparts are located in the green ‘reference’ cluster, meaning that for these ingredients higher dosages usually have greater impact on the parameters measured. The red cluster is populated with several proteins and more specifically animal proteins with good warm gelling and emulsifying properties such as globin, blood plasma, and caseinate ,25,26,27Potato starch and pea fiber at HD also show similar behavior compared to proteins and carrageenans, most likely because starch gelatinization during heating results in inclusion of water and increase of product structure. Potato starch has some advantages compared to other native starches, such as low gelatinization temperature (60–65 °C), high water binding capacity, and high viscosity. The combination of these attributes makes it an attractive ingredient for meat processing .G′-FLM) and structure strength (LVR-FLM) of the raw batter are increased, while on the other hand, a rather strong negative impact on the formation of the liver network during heating is observed (G′ max at 76 °C hold-FLM).The blue cluster mainly contains ingredients such as gums and fibers with a soluble component. Most of those ingredients are present in the cluster both at LD and HD, indicating that small dosages already influence product quality. On the other hand, ingredients at HD have a more pronounced effect on certain parameters  but this does not result into their classification in another cluster, suggesting that the impact of increased dosage on product parameters is rather limited. Gums and fibers with a large proportion of soluble components are characterized by a thickening or weak gelling character, and therefore greatly improve water-binding capacity. These properties result from an equilibrium between water–gum interactions and intermolecular forces , which leads to the formation of aggregates or 3D matrix structures ,12. AccoG′ max at 76 °C hold-FLM). Several authors investigated the impact of alginate on structure and texture of meat products and protein gels. Sarteshnizi, Hosseini, Mousavi Khaneghah and Narges [G′ end cool-FLM was much lower compared to that of the blank.The impact on structural parameters and protein network formation by ingredients in the blue cluster is described by several other authors. Whiting  stated td Narges  suggested Narges  found a G′ max at 76 °C hold-LMM and FLM). According to the authors, this was related to the presence of xanthan in the interstitial spaces of the matrix, where its high molecular weight probably hindered formation of the protein matrix. Also, its construction of large cavities in the network could be linked to the impact on structure. On the other hand Ramírez, Barrera, Morales and Vázquez [This cluster is solely occupied by xanthan at both dosages. As seen in  Vázquez  attributG′ end hold 76 °C- for LMM and FLM, respectively. Despite the impact on product structure formation during processing, hardness of both LMM and FLM is not greatly affected, in contrast to xanthan (orange cluster). Although both orange and purple clusters exhibit ingredient x dosage combinations with divergent behavior, the developed model systems can still distinguish them. The intrinsic characteristics of psyllium, such as its extremely high water-binding and gel properties could explain the position of this ingredient in the purple cluster [The purple cluster contains only two ingredient x dosage combinations: guar and the mixture bamboo and psyllium both at HD. The main reason only few ingredients are present in this cluster is, like in the orange cluster, the rather extreme behavior of the samples as seen in  cluster .The developed toolbox consisting of a lean meat and fatty liver-based model system was a trustworthy measuring instrument to obtain standardized and comparable data concerning intrinsic ingredient functionalities. Hierarchical cluster analysis could create an efficient overview of ‘ingredient x dosage combinations’ based upon similar behavior, rather than product class, while maintaining a great distinctive power. It revealed 5 main ingredient clusters, each with their own unique characteristics. This new approach will help the meat industry and its partners in making the right choice of ingredient for specific meat applications based on scientific, standardized, and comparable data. Ingredient manufacturers and suppliers can also call on this screening tool to reveal the intrinsic characteristics of newly developed ingredients and compare them with what is available in the market. In future work, the obtained knowledge on model system level will be translated to several types of industrial meat products. The parameters measured in the model systems will be linked to quality characteristics of meat products on industrial scale.


Lasiodiplodia has been reported from several grape growing regions and is considered as one of the fastest wood colonizers, causing Botryosphaeria dieback. The aim of this study was to (i) evaluate the efficacy of Esquive®, a biocontrol agent, on vineyard pruning wound protection, applied single or, in a combined protection strategy with a new site-targeted copper-based treatment (LC2017), and (ii) compare their efficacy with chemical protection provided by the commercially available product, Tessior®. For two seasons, protectants were applied onto pruning wounds, while LC2017 was applied throughout the season according to the manufacturer’s instructions. Pruning wounds of two different cultivars were inoculated with three isolates of Lasiodiplodia spp. Efficacy of the wound protectants, varied between both years of the assay and according to the cultivar studied but were able to control the pathogen to some extent. The application of LC2017 did not show clear evidence of improving the control obtained by the sole application of the other products tested. Nevertheless, LC2017 showed a fungistatic effect against Lasiodiplodia spp., in vitro, and has previously shown an elicitor effect against grapevine trunk diseases. Therefore, this combination of two protection strategies may constitute a promising long-term approach to mitigate the impact of Botryosphaeria dieback.The genus  Lasiodiplodia spp. and Neofusicoccum spp. were previously proven as being amongst the fastest wood colonizing genera, and therefore considered as some of the most virulent GTD fungi  × 100  × 100 , where AThe assay was conducted between 2019 and 2020, on an experimental vineyard located at Instituto Superior de Agronomia (ISA) (38°42′33.5″ N 9°11′15.8″ W) Lisbon, Portugal, planted in 1998. Two cultivars were used for this assay, Touriga Nacional (TN) and Cabernet Sauvignon (CS), both grafted onto 140 Ruggeri. Vines were trained as bilateral cordons. Traditional cultural practices in the vineyard were kept throughout the whole assay, and disease management followed an IPM. The products applied were selected with the care of not containing any substance that could casually interfere with the assay.L. theobromae and one L. mediterranea isolates were used for this assay . Lasiodiplodia theobromae isolates Bt105 and LA-SOL3, were collected in Portugal and Peru, respectively, and were stored at the culture collection of Instituto Superior de Agronomia. Both were isolated from grapevine wood showing symptoms of cankers and wood necrosis. The L. mediterranea isolate used is from the CBS culture collection from the Westerdijk Fungal Biodiversity Institute, in Utrecht, Netherlands, with the accession CBS 124060. Although L. mediterranea is currently not reported in Portugal, it has been previously reported in other European countries and so, one isolate of this species was also included in this study, not only for comparison but also to investigate the efficacy of the studied products towards this species. Isolates were maintained in PDA and transferred to Petri dishes with PDA to promote colony growth. Cultures were incubated at 25 °C in complete darkness for 8 days. After incubation, cultures were plated onto 6 mm Petri dishes containing 2% water agar with autoclaved pine needles (Pinus pinea) and incubated at 25 °C under fluorescent light for a 12 h photoperiod, to pycnidia sporulation [5 spores/mL with the use of a hemocytometer .Two is assay . Lasiodirulation ,77,78. OProducts used in this assay can be found in ®  to prevent dehydration and promote spore germination. Pruning and artificial inoculation were performed on the 14 and 15 February 2019, and on the 20 and 21 February 2020, during the winter dormancy, taking into consideration that all the procedures were made during favorable meteorological conditions, namely cloudy and humid, but avoiding rain periods. The same precautions were taken for the remaining applications with LC2017, with the consideration of also avoiding days with strong winds to minimize spray drift.For both seasons, immediately after harvest, one application of LC2017 was carried out as recommended by the manufacturer . HarvestTrichoderma spp. colonies  was used to extract genomic DNA from 8-day-old cultures grown in PDA and incubated at 25 °C, in the dark, following the manufacturer’s instructions. The identity of Lasiodiplodia theobromae and L. mediterranea was confirmed by sequencing part of the translation elongation factor 1α gene (tef1-α) by using the primers EF1-688F and EF1–1251R [T. atroviride strain I-1237 was confirmed by sequencing the internal transcribed spacer region (ITS) using the universal primers ITS5 and ITS4 [For both years, canes were recovered after harvest, during the month of October, and stored in a cold chamber (4 °C) until further processing. For pathogen re-isolation, the bark of each cane was removed, and a sample was collected from about 1 cm below the pruning wound . Four picolonies . A repreF1–1251R , while Tand ITS4 . AmplifiFor both years of the experiment, daily temperature and rainfall were obtained from the Portuguese Institute for Sea and Atmosphere (IPMA—Instituto do Mar e da Atmosfera). These data were collected on the Lisbon reference meteorological station, which is located approximately 7 km from the vineyard tested.www.r-project.org (accessed on 30 September 2021)). The experimental data for both in vitro assays  were compared using an analysis of variance (ANOVA) followed by a Tukey’s test (p = 0.05). Prior to this analysis, Levene’s test was performed in order to verify the homogeneity of variance. Results of the dual culture antagonism assay were plotted using the R package ggplot2. The efficacy of the wound protectants was calculated as the mean percentage recovery (MPR) of the isolates under study. Normality and homogeneity of variance were tested using Levene’s test and when necessary, data were transformed into the arcsine of the square root of the proportion to verify the assumption of homogeneity of variance. For both years and cultivars, an ANOVA was used to compare the differences in the mean percentage of recovery (MPR). The means were compared using Tukey’s test at the 5% significance level (p = 0.05). The mean percentage of disease control was also calculated according to Sosnowski et al. [All statistical analyses were performed using the R program (i et al. ,50 and Mi et al. , using ti et al. .