In Australia, the New South Wales (NSW) State Insurance Regulatory Authority has been continuously developing and implementing clinical practice guidelines to address the health and economic burden from whiplash associated disorders (WAD). Despite this, it is uncertain the extent to which the guidelines are followed. This study aimed to determine insurer and health professional compliance with recommendations of the 2014 NSW clinical practice guidelines for the management of acute WAD; and explore factors related to adherence.This was an observational study involving an audit of 288 randomly-selected claimant files from 4 insurance providers in NSW, Australia between March and October 2016. Data extracted included demographic, claim and injury details, use of health services, and insurer and health professional practices related to the guidelines. Analyses involved descriptive statistics and correlation analysis.p < 0.01).Median time for general practitioner medical consultation was 4 days post-injury and 25 days for physical treatment (e.g. physiotherapy). Rates of x-ray investigations were low (21.5%) and most patients (90%) were given active treatments in line with the guideline recommendations. The frequency of other practices recommended by the guidelines suggested lower guideline adherence in some areas such as; using the Quebec Task Force classification (19.9%); not using specialised imaging for WAD grades I and II ; not using routine passive treatments ; and assessing risk of non-recovery using relevant prognostic tools . Over half of the claimants (59.0%) were referred to other professionals at 9–12 weeks post-injury, among which 31.2% were to psychologists and 68.8% to specialists . Legal representation and lodgment of full claim were associated with increased number of medical visits and imaging  contains supplementary material, which is available to authorized users. In primary musculoskeletal health care, many elements of practice have been identified as being ineffective and costly. Such practices include overuse of imaging and overuse of passive interventions –5. AccorImplementation strategies for musculoskeletal guidelines have shown positive effects on practices such as referral for imaging and provision of active treatments. Requests for x-rays have reduced with strategies such as distribution of educational materials, educational meetings and reminder messages –24. .HeaIn New South Wales (NSW) Australia, clinical practice guidelines for whiplash associated disorders (WAD) have been developed, updated and implemented over the past 17 years by the state insurance regulator, State Insurance Regulatory Authority (SIRA) –29. WAD Consistent with results of studies in other musculoskeletal conditions, implementation strategies for WAD guidelines to date have resulted to varying success in changing professional practice of insurers and health professionals. Awareness of and compliance with the guidelines have increased among insurance staff and health professionals have improved provision of active treatments, reassurance and classification of WAD following an educational session led by opinion leaders –37. HoweFull implementation of guidelines typically takes 3 years  and the In NSW Australia, submission of claim-related documentation such as claim forms (e.g. accident notification form (ANF), personal injury claim form (PICF)) and physical treatment request forms (notification of commencement (NOC) form, review form) are the primary mechanisms for communicating details of management to insurers and accessing treatments after WAD. A systematic evaluation of claim-related documentation provides a picture of the extent to which insurers and health professionals are implementing the WAD guidelines. An audit of claim-related documentation will also allow for exploration of other factors that could influence change in clinical practice. Factors such as legal representation, compensation and claim type have been associated with increased health care utilisation following a motor vehicle crash (MVC) –46. FurtThis study therefore aimed to determine compliance of insurers and health professionals with the recommendations of the 2014 clinical practice guidelines for WAD and examine whether changes in professional practice have been sustained over time in light of previous investigations of WAD guideline implementation. In addition, this study aimed to explore claim-related factors associated with guideline compliance. Knowledge of compliance with guidelines in practice will form the basis for improving injury management processes and informing future implementation strategies.This was an observational study that involved an audit of claimant files. Ethics approval was given by the Northern Sydney Local Health District Human Research Ethics Committee (LNR/16/HAWKE/78).Four compulsory third party insurers in NSW, Australia agreed to participate in this study. New South Wales operates under a common law fault-based scheme where injury management costs are covered by compulsory third party (CTP) insurance. CTP insurance is underwritten by private insurance companies regulated by a government authority (SIRA). To be eligible for benefits people with WAD can submit a claim either within 28 days (ANF) or within 6 months (PICF) of the accident . ANF allA random sample of 288 files, selected based on the relative market share of the insurers, was deemed to be representative. The sample was calculated from 1146 new claims submitted between 1 August 2015 and 15 November 2015, more than 6 months from the release of the most recent version of the guidelines in December 2014. This was necessary to provide adequate time for implementation and uptake of the recommendations. Inclusion criteria were; (1) whiplash as primary injury, (2) age of claimant > 17, and (3) no fatality reported.Primary sources of data were claim forms , medical certificate, NOC and review forms, and insurer file notes. The ANF and PICF, accompanied by a medical certificate, outline demographic and injury details, results of general medical practitioner (GP) assessment, request for imaging, referral to physical treatment  and medications . The NOCFour researchers attended the participating insurer offices to extract data using a standard form. The standard form was a password-encrypted Excel spreadsheet created by the researchers to obtain relevant information to address the aims of the study. Another researcher randomly extracted data from 20% of all files to compare for agreement with the data extracted by other researchers. Fortnightly meetings were held to resolve disagreements and ensure consistency of data extraction.Data extracted included demographic, claim and injury details, use of health services, and health professional and insurer practices. Demographic information included age, gender, employment status and postcode. Claim details included date of claim lodgement, claim status, claim type  and legal representation. Injury details included accident date and road user type. Use of health services provided information about access to recommended treatments and performance of the scheme and included timing and numbers of medical and physical treatments. Insurer and health professional practice related to compliance with guideline recommendations on classification, use of radiology, prognosis, treatment and referral  WAD grade , Numeric Rating Scale (NRS)), Neck Disability Index (NDI) , Impact Data were analysed using descriptive statistics ; frequencies (n(%))). Data for treatment were analysed across the time points; commencement of physical treatment, 6, 12 and 26 weeks post-injury, pertaining to acute, sub-acute and chronic periods of management, respectively. The time points were used as basis to determine whether the number of treatment sessions and provision of adjunctive treatments were tapering over time, for claims with multiple reviews. The researchers made judgements about WAD grade, indications for imaging and appropriateness of referral based on available file information.Association of demographic factors  and claim characteristics  with measures of guideline compliance  was determined using correlation analysis. The Shapiro-Wilk statistic revealed that the continuous data were not normally distributed; hence, non-parametric tests of association were used. Correlation coefficients were interpreted as: 0.00 to 0.29, small; 0.30 to 0.49, medium; and 0.50 to 1.0, strong correlation . StatistData extraction occurred from March until October 2016 . Health professionals provided WAD grade in 13.0% of the files (n = 35/288). The most frequently reported diagnostic label was ‘whiplash injury’ . Use of patho-anatomical terms was observed in 26.7% of the files (n = 77/288).Insurers requested WAD grade in 6.9% of the files (n = 167/288) (Table Claimants received at least one form of imaging in 57.9% of the files n = 167/88 (Tablen = 49/62) of the files.The median time to insurer response to x-ray requests was 22 days. Five files (8.1%) had documented approval of request to rule out cervical fracture and 5 requests (8.1%) were declined for lack of clinical indications. There was no record of insurer response to the request in 79.0% (n = 81/288). For x-rays conducted outside of emergency department (ED), 90.3% of the requests were made by the GP (n = 56/62) due to neck pain, tenderness, loss of motion .X-rays were conducted for 28.1% of the claimants (n = 47/132) to clarify diagnosis. Twenty-one (15.9%) requests were declined for lack of clinical indications. There was no record of insurer response to the request in 34.1% of the files (n = 45/132).The median time to insurer response to specialised imaging requests was 15 days. Approval was given for 35.6% of the requests , with 80 claimants receiving at least one specialised imaging and 61 receiving more than one specialised imaging. The majority of specialised imaging was done outside of ED  and requested by GPs  for reasons such as neck pain, tenderness, loss of motion . Magnetic resonance imaging (MRI) was most commonly requested . The majority of those that received specialised imaging were claimants classified as WAD Grade II  of specialist imaging requests were considered compliant with the guidelines given presence of symptoms requiring further imaging to rule out possible neurological pathology.Based on the review of all files and researcher judgment, 31.8%  (Table n = 4/288) to 36.5%  of the files  were referred for physical treatment, of which 237 (94.8%) had documented evidence of receiving treatment. The most common health professionals referred to were physiotherapists . Approximately 13% (n = 36/288) of claimants received physical treatment 1 week post-injury. The highest proportion of claimants receiving physical treatment was at 9 weeks post-injury   at 26 weeks.The median time to GP consult was 4 days after injury and 25 days for physical treatment. The average number of GP consults was 5 sessions and 15 sessions for physical treatment. The majority of claimants (n = 187/201) of physical treatment requests to promote early treatment, self-management and progress to functional recovery. Physical treatment request was denied in 5% (n = 9/201) of the files for reasons including lack of evidence for physical benefit.The median time to insurer response to physical treatment requests was 20 days after receipt of the NOC forms. Insurers approved 93%  and the majority received a combination of first-line and adjunctive treatments  , advice  and manual therapy  (Table Few people with WAD received first-line treatments only (n = 92/201) of claimants had one session of physical treatment per week and 45.8% (n = 92/201) had 2 sessions per week  were referred to other professionals  to confirm diagnosis, assess current status, review appropriateness of treatment and provide recommendations on rehabilitation needs. Insurers declined 10% of specialist referrals (n = 11/117) due to lack of indicators for specialist consult.The median time to insurer response to specialist referral was 18 days. Request for specialist referral was approved in 20.5% of the files  were referred to specialists (Table n = 51/117) involved referral to surgical specialists and 20.5% (n = 24/117) to WAD specialists. The main justification for specialist referral was to obtain opinions about diagnosis and management . The median time for referral to specialists was 89 days. Outcomes of referral included clarification of condition  and request for further imaging and tests .One-hundred-seventeen claimants (ts Table . Of thesn = 55/288) would have benefited from WAD specialist consult where no referral was made. These indications were obtained from reports of apparent non-recovery at review and presence of high-risk factors, such as high pain and disability.Based on review of files and researcher judgment, an additional 19.1% of claimants (n = 29/53) to address psychological needs and declined 13.2% of the requests (n = 7/53) due to lack of indicators.The median time to insurer response to psychology referral was 44 days. Insurers approved 54.7% of the referral requests  were referred to psychologists due to presence of anxiety and fear of driving  (Table Fifty-three claimants (%) Table . The medn = 39/53) of the referrals made to psychologists were considered appropriate to address psychological symptoms. There was also a proportion of claimants deemed that would have benefited from psychologist consult due to reported presence of psychological symptoms ; however, no referral was made.Based on review of files and researcher judgment, 73.6% , and between open claim and increased number of GP consults . A small correlation was observed between legal representation and increased number of specialised imaging received  and GP consults , and higher total cost of physical treatment .There was moderate correlation between full claim and increased number of specialised imaging received  or patho-anatomical diagnoses (26.7%), rather than the QTF classification. Further, high rates of MRI were observed despite majority of claimants being classified as Grade II, which is consistent with pursuing a patho-anatomical diagnosis. Use of unnecessary imaging in WAD has also been observed in Victoria, Australia .2% or pa, 58. ThiThe high proportion of claimants receiving passive treatments beyond the acute phase of injury (> 12 weeks) may also reflect an approach that is inconsistent with guideline recommendations. Manual therapy is often provided for people with WAD in other countries , 71, sugFurther, both insurer and health professional practices demonstrate non-compliance with identification of claimants at risk of non-recovery and acting on this with timely and appropriate referral. Health professionals and insurers inconsistently used validated prognostic tools and rarely reported on expectation of recovery. Most referrals were made to surgical specialists, arguably too late (> 13 weeks) to influence the course of recovery as studies on WAD recovery trajectories demonstrate that most recovery occurs within 12 weeks of injury , 77–79. Given that implementation strategies to date  have not been completely successful in promoting compliance with all of the recommendations in the guidelines, alternative strategies may need to be considered. One strategy may be to implement organisational changes such as mandating practice through policy or within insurer claims management processes. Regulatory or legislative changes may also be implemented by the insurance regulator to involve restrictions in payment of treatment requests or having a reasonable cap for number of treatments provided. Evidence in other settings showed that changing policies at the level of the organisation resulted to changes in practice. Reimbursement restriction policies decreased inappropriate medication prescribing , and proAt the level of the insurers, changes in claims management processes may include mandated use of validated risk screening tools, such as the clinical prediction rule for WAD , 83, andProfessional implementation strategies have been effective in instigating practice change; however, there are entrenched behaviours remaining unchanged and appeared to be influenced by scheme and compensation factors. Results of the current study showed that having legal representation and full, ongoing claims were associated with higher number of medical visits, specialised imaging and cost of treatment. Although associations were weak to moderate, these results highlight that organisational and regulatory changes, in addition to professional implementation strategies, may be necessary to change practice.Whilst data were extracted from all available sources, this study was limited by the completeness of the files. The majority of the claimant files were open, ongoing claims at the time of data extraction; hence, some data were not available (e.g. insurer correspondence). Another limitation could be that the files were obtained from new claims submitted approximately 8 months after the release of the most recent WAD guidelines. Insufficient time may have elapsed for the guideline recommendations to be adopted in practice. Additionally, only insurer files were audited; hence, details of specific treatments and management decisions made by health professionals may not have been adequately captured. Lastly, results may not be generalised to practices not operating within the same healthcare and compulsory third party insurance systems as management processes and decisions may differ.In summary, there is evidence of insurer and health professional practices that are compliant with recommendations from WAD guidelines. Claimants receive early access to and approval of appropriate care. However, there is also evidence of practices that are not compliant and might lead to poor health outcomes and greater treatment costs. Alternate implementation strategies may need to be considered to reduce unnecessary imaging and the persistent provision of passive treatments; promote use of validated prognostic tools; and maximise early referral to WAD specialists and psychologists. It appears that multiple strategies involving organisational, regulatory and professional interventions may be necessary to successfully implement the recommendations of the WAD guidelines to change practice, improve scheme performance and ultimately improve outcomes for people with WAD.Additional file 1:Appendix 1. Insurer and health professional data collected based on recommendations of the guidelines. Appendix 2. Quebec Task Force classification of grades of WAD. Appendix 3. Flow diagram of claimant files included in the study. (DOCX 37 kb)


The protective efficacy of the most promising malaria whole-parasite based vaccine candidates critically depends on the parasite’s potential to migrate in the human host. Key components of the parasite motility machinery  have been identified, however the regulation of this machinery is an unknown process.In vitro, their motility; their adherence capacity, movement pattern and velocity during forward locomotion.In vitro microscopic live imaging of parasites in different formulations was performed and analysed, with the quantitative analysis software SMOOTIn vitro enabled the detailed analysis of the regulation of the motility machinery of Plasmodium berghei in response to specific (macro)molecules in the formulation. Albumin acted as an essential supplement to induce parasite attachment and movement. Glucose, salts and other whole serum components further increased the attachment rate and regulated the velocity of the movement.SMOOTBased on the findings can be concluded that a complex interplay of albumin, glucose and certain salts and amino acids regulates parasite motility. Insights in parasite motility regulation by supplements in solution potentially provide a way to optimize the whole-parasite malaria vaccine formulation.The online version of this article (10.1186/s12936-019-2794-y) contains supplementary material, which is available to authorized users. Plasmodium parasites are injected into the skin by a probing Anopheles mosquito in the highly motile sporozoite (spz) stage [In 2017, an estimated 219 million cases of malaria occurred worldwide leading to nearly half a million deaths, which makes malaria the most deadly parasitic disease worldwide . Infection with malaria is initiated when z) stage . NeverthAfter intradermal deposition, spz rely on their own adhesion capacity and actin/myosin-based motility machinery to migrate. Their adhesion capacity relies on proteins, e.g. the circumsporozoite protein (CSP) and the thrombospondin-related adhesive protein (TRAP), which can form adhesion sites , 5. The Plasmodium berghei and Plasmodium yoelii expressing fluorescent proteins  [To shed light on spz motility, transgenic spz e.g. in; GFP)  have beein; GFP) , 17. Thein; GFP) –20, possin; GFP) , 21. Stuin; GFP) , 23. Howin; GFP) –26. MoreClassically, gliding assays are used as the gold standard for assessing spz motility in vitro , 28, butPlasmodium berghei-mCherry when augmented with a quantitative analysis tool have the potential to provide a means to assess the regulation of P. berghei adhesion and locomotion by formulation composition. To illustrate the potential of this concept, the custom image analysis software called SMOOTIn vitro (Sporozoite Motility Orienting and Organizing Tool) has been developed and used to study the effect of a systematic variation in formulations: PBS, HBSS and RPMI, enriched with either albumin or whole serum.In vitro microscopic imaging studies with Plasmodium berghei spz  which contain the fusion gene mcherry under control of the strong hsp70 promoter integrated into the neutral 230p gene locus (PBANKA_0306000) were obtained by microsurgical dissection of the salivary glands of infected female Anopheles stephensi mosquitoes. To allow the spz to mature dissections were performed 21–24 days following infection of the mosquitoes. During this period mosquitoes were kept at a temperature of 21 °C and 80% humidity. The salivary glands were collected and crushed in the different formulations mentioned in the next section. The free spz were counted in a Bürker counting chamber using phase-contrast microscopy. On average, dissection yielded 55 k spz/mosquito. The mosquitoes and the spz were kept on ice (0–4 °C) for a maximum of 3 h until use.Mosquitoes were infected by feeding on infected mice  as described previously . The aniTo investigate which components are needed to induce motility of spz, the mosquitoes were dissected and the obtained salivary glands were crushed in eleven different formulations: 1) Phosphate buffered saline which contains potassium, sodium, chloride and phosphate , (2) Hanks’ balanced salt solution which contains besides the salts present in PBS also glucose, calcium, magnesium, sulphate and bicarbonate , (3) Roswell Park Memorial Institute medium  which contains besides the components of HBSS also amino acids and vitamins, (4) PBS enriched with 3.5 mg/ml bovine serum albumin , (5) HBSS + 3.5 mg/ml BSA, (6) RPMI + 3.5 mg/ml BSA, (7) PBS enriched with 10% fetal bovine serum , (8) HBSS + 10% FBS, (9) RPMI + 10% FBS, (10) RPMI without amino acids (Caisson Labs) + 3.5 mg/ml BSA and (11) RPMI without glucose (Life Technologies Inc.) + 3 mg/ml BSA. All eleven formulations had a physiological pH of 7.0–7.5 and a viscosity of < 1.1 cP. For imaging of the spz, 10 µl of the spz solution was pipetted on the cover slip of a confocal dish without any precoating , covered with another cover slip  and imaged within 45 min. The set-up is schematically depicted in Additional file  PhosphatImages of the spz were taken on a Leica TCS  SP5 or SP8X WLL (white light laser) microscope . The spz expressed mCherry which was excited at 587 nm and the emission was collected between 600 and 650 nm. The movies were recorded with a frame rate of 35 frames per minute, 400 frames per movie, three movies per condition. The resolution of the images was set at 1024 × 1024 pixels. For imaging of the spz a 40× objective (Leica HCX PL APO CS 40×/1.25–0.75 na OIL) was used and the experiments were performed at room temperature, unless otherwise stated. The movies were recorded using the Leica software .In vitro, an in-house developed graphical user interface (GUI), written in the MATLAB programming environment . Via SMOOTIn vitro analysis the spz could be segmented per movie frame, based on their fluorescence signal intensity, size and crescent shape by applying a binary threshold, a median filter, skeleton adjustment and spot removal. Colliding spz and spz circling on the edge of the field of view were excluded, since the locations of these spz on the consecutive frames cannot be stitched together in a reliable way. The value of these segmentation parameters can be set in the GUI. The direct visual feedback provided by the GUI during the setting of the parameters was used to find the optimal segmentation settings. Over time the median pixel locations of segmented spz visible on different frames could be connected into full spz tracks, which were subsequently separated in segments based on changes in movement pattern. The movement pattern of the spz was classified per segment as floating, stationary or circling. The velocity and turn angle at frame level, the turn direction (clockwise (CW) or counter-CW (CCW)) at segment level and the length of the travelled path (expressed as number of frames and displacement per frame) at track level of the circling spz were determined. On average ~ 100 spz were analysed per condition.Maximum projections of the recorded microscopy movies were generated using Fiji software . The movt test depending on the nature of the variables tested . p-values < 0.05 were considered significant. All statistical tests were performed by SPSS Statistics .Statistical analysis consisted of the One-way ANOVA, the Mann–Whitney U test and the independent sample In vitro was able to discern three main spz movement patterns in the microscopic spz movies: floating (red), stationary (green) and circling  was studied: PBS, HBSS and RPMI (f1–3), which were enriched with either 3.5 mg/ml purified BSA (f4–6) or 10% FBS which contains amongst other things ~ 3.5 mg/ml BSA (f6–9). Changes in the formulation yielded a direct effect on the spz movement patterns. Typically, in formulations without BSA or FBS (f1–3) and also in the condition PBS + BSA (f4), most spz were floating. An example of such an effect is shown in Fig. Using automated SMOOTQuantitative analysis confirmed that the movement behaviour of spz was significantly different between the conditions with and without BSA and FBS , many other components like salts, glucose, vitamins and cholesterol. Enrichment of any of the formulations with FBS induced circling of 60–80% of the spz. Significantly more spz were circling in the presence of FBS compared to BSA when it was added to PBS or HBSS , between these formulations striking variations could be observed in the distribution of the velocities measured Fig. c: 5–9. CIn vitro) enabled a quantitative analysis of spz motility by describing their (1) attachment rate, (2) movement pattern distribution and (3) velocity distribution. Because SMOOTIn vitro enabled to tease apart spz motility in tracks, segments and frames, the different motility parameters could be individually dissected and subtle differences could be quantified. In this way has been confirmed that albumin is essential to induce spz attachment and movement. Glucose, salts, amino acids and vitamins, components in RPMI, further increased the attachment rate and the percentage of moving spz, whereas the supplements present in whole serum regulated the velocity of the spz movement. Combined, these findings indicate that the regulation of spz motility is a complex interplay of spz with different macromolecules and salts.The availability of a dedicated analysis tool  motility inhibiting drugs at different concentrations. Potentially, motility analysis with SMOOTIn vitro could be performed as an additional assay to previously described high-throughput screening methods [The three in vitro movement patterns, floating, stationary (fully attached and waving) and circling (> 95% CCW), which were observed for  reports , 28, 32.erformed , 33. Parerformed . The remSpz attachment and movement are two distinguishable, but likely related steps in spz motility. Both appear to be initiated by external stimuli transferred by internal signalling cascades. Hegge et al. have described spz adherence and movement as a four step procedure : step 1 Interestingly, albumin and glucose do not seem to be the sole supplements regulating spz motility. In addition, supplements present in RPMI  act as stimuli for spz attachment and subsequent moving. As spz can also use glutamate or glutamine to enter the Krebs cycle to produce energy and the availability of these compounds influences spz motility , 33, thiThe available (macro)molecules did not only regulate the occurrence of spz adherence and moving, but also the velocity of their forward locomotion. Besides attachment to the surface via adhesive proteins, also detachment by the turnover of the adhesion sites is essential to allow the spz to move forwards at a certain speed , 36. ThiIn vitro analysis of the effect of different potential stimuli and inhibitors has the potential to provide insight in the biological mechanisms behind spz motility. A recently developed fluorescent labelling approach for non-fluorescent parasites provides the opportunity to study the motility of spz in vaccine preparations or isolated in the field [In vitro can be adapted to study the motility of malaria parasites in other developmental stages (e.g. ookinetes) if the segmentation and stitching parameters would be adjusted and validated to select the parasite.The comparison between the attachment rate, movement pattern distribution and velocity distribution induced by the different formulations suggested two different trends: (1) whole serum both increased the attachment rate, the percentage of circling spz and their velocity compared to albumin and (2) RPMI increased the attachment rate and the percentage of circling spz, but decreased their velocity compared to PBS and HBSS. Combined these trends indicate that spz motility is dependent on regulators inducing the right balance between strong attachment and fast detachment. The SMOOThe field . PotentiDespite the regulating role of the different supplements on spz motility, PfSPZ vaccines currently are formulated in PBS with albumin only . It is nIn vitro allowed for a quantitative assessment of the different conditions studied and with that helped isolate the supplements that were key for spz motility. This spz motility analysis tool can be of future use to gain insight in the mechanisms behind spz migratory behaviour, to determine the most optimal vaccine formulation, to study the effect of radiation and chemical attenuation of spz and to screen potential spz motility inhibiting drugs.This initial in vitro study indicated that (macro)molecules effect spz motility by acting as regulators of adherence to the surface, induction of forward locomotion and the velocity of this movement. SMOOTAdditional file 1: Figure S1. Spz motility analysis tool. A) The Plasmodium berghei spz were obtained by manual dissection and crushing of the salivary glands of infected Anopheles stephensi mosquitoes. B) The behaviour of spz in different types of media was studied. C) The spz were imaged by confocal microscopy and analyzed using the software SMOOT.Additional file 2: Table S1. (Counter) clockwise movement. A) Distribution of clockwise (CW) and counter-clockwise (CCW) turning spz for the pooled dataset. B) Distribution of CW and CCW turning spz per condition.Additional file 3: Figure S2. Sporozoites circling diameter. The average diameter of the circles of turning sporozoites in RPMI and RPMI without glucose (both enriched with BSA) were 12.3 µm and 12.2 µm respectively. The average diameter of the circles of turning sporozoites in RPMI without amino acids and enriched with BSA was significantly smaller: 10.3 µm .


Tumor growth dynamics vary substantially in non-small cell lung cancer (NSCLC). We aimed to develop biomarkers reflecting longitudinal change of radiomic features in NSCLC and evaluate their prognostic power. Fifty-three patients with advanced NSCLC were included. Three primary variables reflecting patterns of longitudinal change were extracted: area under the curve of longitudinal change (AUC1), beta value reflecting slope over time, and AUC2, a value obtained by considering the slope and area over the longitudinal change of features. We constructed models for predicting survival with multivariate cox regression, and identified the performance of these models. AUC2 exhibited an excellent correlation between patterns of longitudinal volume change and a significant difference in overall survival time. Multivariate regression analysis based on cut-off values of radiomic features extracted from baseline CT and AUC2 showed that kurtosis of positive pixel values and surface area from baseline CT, AUC2 of density, skewness of positive pixel values, and entropy at inner portion were associated with overall survival. For the prediction model, the areas under the receiver operating characteristic curve (AUROC) were 0.948 and 0.862 at 1 and 3 years of follow-up, respectively. Longitudinal change of radiomic tumor features may serve as prognostic biomarkers in patients with advanced NSCLC. Cancers have uneven growth patterns, and even within single tumors specific areas may increase in size while other portions remain stable. Intratumoral heterogeneity reflecting heterogeneous biologic phenomenon can affect the rates and patterns of tumor growth2. With the development of targeted therapy and immunotherapy, tumor cells remain sensitive to targeted therapy beyond progressive disease (PD) according to Response Evaluation Criteria in Solid Tumors (RECIST), while pseudoprogression occurs in patients undergoing immunotherapy due to infiltration of T cells and macrophages3. On the other hand, targeted therapy or immunotherapy is used continuously during tumor progression4, and as assessment of a therapeutic response is usually made between two time points, it may not capture the full spectrum of therapeutic response and could be insufficient in the era of personalized cancer treatment5.Accurate radiological assessment for predicting therapeutic responses and clinical outcomes of non-small cell lung cancer (NSCLC) is important in clinical practice and drug development trials6. Recently, depth of response (DepoR), which indicates the maximum tumor shrinkage from baseline of a target lesion, has begun to be explored as a predictor of long-term treatment outcome7. However, recent studies suggest that DepoR should not be used a surrogate of benefit in advanced epidermal growth factor receptor (EGFR)-mutant lung cancer8. Therefore, parameters such as continuous variables reflecting intratumoral heterogeneity and genomic alterations are needed to assess therapeutic response and clinical outcomes.Rapid reduction of tumor size is traditionally considered to be associated with better outcomes10. Changes in quantitative features are considered a biomarker to accurately assess treatment response and clinical outcomes11. Recently, delta-radiomics, a time dependent metric comprised of quantitative features extracted from medical images acquired during the course of treatment has been considered as an emerging biomarker for assessing therapeutic responses and clinical outcomes12. We hypothesized that variables capable of reflecting various longitudinal changes in quantitative image features extracted from serial imaging over the course of treatment would be capable of superior prognosis prediction in NSCLC patients. The purpose of this study was to develop biomarkers based on longitudinal changes in quantitative radiomic features in patients with advanced NSCLC, and assess the prognostic power of these biomarkers.Quantitative imaging features may provide comprehensive information on tumor phenotypes and microenvironementThis study included 53 patients with stage IV NSCLC . The histologic types of all patients were adenocarcinoma. Thirty-four patients (64.2%) were never smokers, 62.3% had exon 19 deletion, and 56.6% were treated with EGFR TKIs as a first-line of therapy. Forty (75.5%) patients had metastasis to the central nervous system (CNS), 27 of whom underwent local treatment. Among 53 patients, 27 (50.9%) died. The number of target lesions was 1 in 26 patients (49.0%), 2 in 15 (28.3%), 3 in 10 (18.9%), and 4 in 2 (3.8%). The mean CT follow-up period from baseline to PD was 28 ± 15 months. The characteristics of patients with advanced NSCLCs are presented in Table p < 0.000). Furthermore, AUC2 reflecting under the area and slope during the follow-up period was well correlated with the six patterns of volume change  as well as beta value .For the prediction of overall survival time, we calculated the cutoff of AUC1, AUC2, and beta value based on volume. When we selected cutoff values of 1.601 for AUC1, −0.738 for AUC2, and −0.176 for the beta value, the AUC2 and beta values showed significant differences in overall survival time . Six of the 18 features were selected by stepwise selection. Kurtosis of positive pixel values  and surface area  on baseline CT, and AUC2 of density , skewness of positive pixel values  and entropy at inner portion  were associated with overall survival in multivariate cox analysis in patients with advanced NSCLC  inhibitor or a programed cell death-1 (PD-1) inhibitor5. On the other hand, another study reported that DepoR does not correlate with clinical outcome8. Therefore, an approach based on a single marker is not sensitive to assessment of therapeutic response due to phenotypic and genomic heterogeneity between lesions, even within an individual tumor18.As the use of targeted molecular therapy and immunotherapy increases in the era of personalized cancer treatment, morphological changes may be more important than changes in tumor size, and increases in the size of new lesions before dramatic shrinkage may also occur in relation to the immune response19. The use of radiomics in lung cancer studies is focused on patient outcomes, nodule discrimination, and assessment of genetic mutations20. Whereas most radiomics studies are performed using pretreatment images, the concept of delta-radiomics was introduced to analyze quantitative features from images acquired over the course of treatment by adding a time component to existing radiomic features12. Delta-radiomics may be used as indicator to predict response in colorectal liver metastasis, metastatic lung cancer, esophageal cancer, and NSCLC25. Previous studies have shown that tumor volume is a key predictor reflecting histological tumor aggressiveness and poor prognosis in patients with NSCLC27. Volumetric measurements that are able to capture tumor changes along the z-axis may be useful for more sensitively and specifically monitoring disease change over time30. Therefore, in our study, we examined longitudinal changes in tumor volume. We measured additional variables reflecting longitudinal change, and from those variable calculated corresponding AUC1 values reflecting the area of volume change, beta value reflecting slope of change, and AUC2 reflecting area and slope. AUC2 exhibited a good correlation with curve pattern  and beta value , whereas the AUC2 of tumor volume was associated with overall survival (p = 0.035). Thus, AUC2 may be a new biomarker that reflects longitudinal changes in lesions and predicts overall survival time and treatment response.Radiomics is defined as quantitative mapping that converts medial images to automatically extracted quantitative features and provides information related to the comprehensive pathophysiology of a tumor23. However, radiomic features can provide more comprehensive information about tumor makeup. A study showed that a novel set of quantitative image features, based on heterogeneities of tumor physiology, was helpful for early prediction of treatment outcome31. Therefore, we obtained AUC2 values for 23 quantitative features, including volume, that were previously reported to be associated with lung cancer37. We examined the association between these 23 pretreatment quantitative features and 23 AUC2s obtained from serial images with overall survival. The result of our study showed that kurtosis of positive pixel values (p = <0.000) and surface area (p = 0.001) on baseline CT, and AUC2 of density (p < 0.000), skewness of positive pixel values (p = 0.010), and entropy at inner portion (p = 0.001) were all associated with overall survival according to a multivariate analysis in patients with advanced NSCLC.Size and volume of a tumor do not reflect changes in tumor heterogeneity and genetic profiles27. In patients with a gastrointestinal stromal tumor, treatment with imatinib results in a decrease in tumor density such that the appearance becomes homogenous on CT, even though the decrease in tumor size is minimal at an early posttreatment time point38. In our study with advanced NSCLC, density change was associated with increased survival, and it has been suggested that increased homogeneity during the course of treatment is related with therapeutic response and prognosis. Two studies reported that a reduction of entropy reflecting tumor heterogeneity as a parameter to evaluate treatment response in colorectal liver metastases and renal cell carcinoma25. In our study, we found that the AUC2 of entropy at inner portion was successful for predicting overall survival time in advanced NSCLC. In addition, the AUC2 of the skewness of positive pixel value was found to be predictor of overall survival. Skewness is the distribution pattern of CT attenuation values, and decreased skewness reflects decreased enhancement due to decreased neovascularization during the course of treatment34. Therefore, based on our results, changes in tumor heterogeneity were related to overall survival time.Targeted therapies developed in recent years can cause tumor necrosis, hemorrhage, or cavitation, and radiomic features based on histograms may be useful for predicting these pathologic response39. Irregularity of tumor shape reflecting non-uniform growth of tumors is associated with worse overall survival in patients with lung squamous cell carcinoma and lung adenocarcinoma40. In our study, surface area on baseline CT was associated with overall survival time in patients with advanced lung cancer, and morphologic features of tumor were considered important predictors related to clinical outcomes.Morphological features reflecting the physical characteristics of tumors are important features related to aggressiveness42. In order to minimize this effect, we excluded patients who had severe respiratory difference in chest CTs or had concurrent disease such as effusion. Fourth, we selected target lesions according to RECIST (version 1.1) criteria. However, current RECIST guidelines may not reflect the overall body tumor load in patients with metastatic cancer. In addition, selection of target lesions is crucial and the result of response classification could be changed by target lesion selection44. The final limitation of our study was that the ROIs were drawn manually by a single operator. As tumor segmentation is the most critical process, and it is well recognized that interoperator variability of manual segmentation of lesion is high9.This study had several limitations. First, the sample size of the present study was small, and our study included patients in various treatment course. However, inclusion of patients with various treatment course may provide a better, more comprehensive assessment of overall survival of patients with advanced lung cancer. A second limitation of this study was that we did not externally validate our results. Therefore, subsequent studies with a large population and external validation are necessary for validation of our results. Third, tumor volume and radiomic features are susceptible to respiration due to collapse and stretching of the tumor and peritumoral lung parenchymaOur study showed that AUC2, a value that reflects the slope and area of longitudinal change calculated from radiomic measurements, is associated with patient survival. Quantitative analysis is a noninvasive technique reflecting comprehensive information such as tumor heterogeneity and genetic profile. In addition, changes in these radiomics features during serial follow-up may provide comprehensive information about response to treatment and prognosis. Therefore, the results of our study suggest that longitudinal change of radiomic tumor features may be useful as prognostic biomarkers in patients with advanced NSCLC.EGFR mutation treated with a tyrosine kinase inhibitor (TKI) and who had been evaluated for therapeutic response on computed tomography (CT) until PD on treatment. Treatment response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST version 1.1). There was no change in TKI type during the course of treatment. Twenty-seven patients were excluded from our study based on the following exclusion criteria: (1) patients who underwent surgical resection (n = 9), (2) patients without stage IV disease (n = 1), and (3) patient without available serial CTs (n = 17). Thus, a total of 53 patients with advanced NSCLC were included in this retrospective study. The study was approved by the Institutional Review Board (IRB) of Samsung Medical Center (IRB number 2015-10-108), and the requirement for informed consent was waived.From January 2012 to October 2014, we recruited 80 patients with advanced or recurrent NSCLC with confirmed All helical CT images were obtained with a 64 detector-row  CT scanner using the following parameters: detector collimation, 1.25 or 0.625 mm; field of view, 36 cm; 125 mA; 120 kVp; beam width, 10–20 mm; beam pitch, 1.375–1.5; section thickness 2.5 mm; and matrix, 512 × 512 mm. All patients underwent chest CT at full inspiration through breath hold to minimize the effect of the tumor motion due to breathing. Chest CT scanning was obtained 90 seconds after the administration of contrast material. A total of 1.5 mL/kg (body weight) Iomeron 300  was injected at an infusion rate of 3 mL/s using a power injector . Image data were reconstructed with a soft-tissue algorithm for mediastinal window ranges and a bone algorithm for lung window images. Both mediastinal  and lung  window images were displayed for tumor assessment. Chest CT images were obtained every two cycles (8 weeks) during the course of treatment.Based on RECIST criteria, up to 5 target lesions on baseline and follow-up chest CT were segmented by drawing a region of interest (ROI) that traced the edge of the lesion on all axial images until the entire lesion was covered. Nontarget lesions were ignored for the analysis of tumor change. Quantitative features were computed over an ROI drawn by a radiologist using MRIcro . From the baseline to the PD time point, a total of 161 quantitative CT features from each serial CTs were computed using a MATLAB function designed in house . Of the 161 quantitative CT features, 23 features with a known association with lung cancer were selected and used for our analysis , beta value, and AUC2 . A p value less than 0.05 was considered statistically significant.Analyses were performed using SAS version 9.4  and R 3.3.1  to see the collinearity between the features included in the multivariable cox regression. The VIF of all variables was less than and there was no collinearity. To identify significant features on Cox’s proportional hazard model, we considered stepwise selection with Akaike’s Information Criterion (AIC). For significant features of overall survival, a time-dependent receiver operating characteristic (ROC) curve was constructed, and the area under the ROC curve (AUROC) was calculated.We calculated AUC2 for each of the 23 quantitative features. The cut-off values that best predicted overall survival time for the 23 baseline quantitative features and AUC2 of 23 quantitative features were selected. Univariate and multivariate analyses of the 23 baseline quantitative features with age, sex, ECOG performance state, smoking status, type of EGFR mutation, and AUC2 of the 23 quantitative features were performed using Cox’s proportional hazards model to identify risk factors associated with overall survival. Univariate analysis was used to select 18 features with a Supplementary table


The aim of this study was to evaluate the effect of dietary incorporation of tannin-rich woody species on meat oxidative stability, carcass traits and meat quality in goats.Larrea divaricata and Acacia aroma both at 12.5% in dry matter basis of the diet and a control diet . All feeding diets were iso-protein and iso-energy. Carcass conformation, carcass compactness, carcass fatness and subcutaneous fat deposition were evaluated. Intake, liveweigh, Longissimus thoracis et lumborum muscles of goats were analyzed in order to evaluate quality parameters such as pH value, instrumental color evaluation, water holding capacity, total phenolic content, antioxidant activity, meat oxidative stability and fatty acid profiles in meat.Two tannin-rich species were tested using a three-treatments feeding trial, where treatments consisted of: Feed intake, liveweight gain, carcass, and meat traits did not differ among treatments. Changes in meat lipid profile among treatments were observed for oleic and elaidic acid contents. Meat total phenolic content and antioxidant activity did not differ among treatments; although, meat oxidative status after storage at room temperature, as well as under refrigerated and frozen conditions were different between control and both supplemented groups.Acacia aroma and Larrea divaricata leaves in goat diet enhanced meat oxidative stability. Modulation of the ruminal biohydrogenation of fatty acids produced by condensed tannins of these plant species need to be further investigated.The inclusion of  Goat meat is considered a healthy product and represents an important source of animal protein and iron for human diet . HoweverIn food industry, a regular strategy to prevent or retard food’s oxidation implies the addi tion of antioxidants directly to food formulation or coated containers made with antioxidant agents ,4. On thAcacia cyanophylla, Schinopsis lorentzii, and Ceratonia siliqua reduced the biohydrogenation of polyunsaturated fatty acids (PUFA). On the other hand, supplementing the goat diet with pine bark rich in CT did not find differences in meat oxidative stability against a diet with low tannins input [Caesalpinia paraguariensis, Schinopsis balansae, and Larrea divaricata (LD) were the richest species in CT as native forage. However, LD and Acacia aroma (AA) had the highest biological activity, followed by Prosopis alba. According to their nutritive traits, these species might be also complementary in grass-based ruminant diets [In goats’ production systems of the semiarid Argentine Chaco, woody perennial plants represent an important source of supplementary feed during the dry season when fresh forage availability is limited . Woody pns input . In a prns input . Some spnt diets .Based on the above, the main objective of this work was to evaluate the effect of the inclusion of two native woody species (LD and AA) as a source of CT in the diet for goats, on animal performance, feed intake, carcass traits and meat quality as well as on meat oxidative status and oxidative stability during refrigerated storage.Leaves of LD and AA were collected from native wild plants in the Argentine Dry Chaco  in November 2014. Leaves were harvested and air-dried under shade on a clean plastic. Once leaves were dried, they were easily removed from the branches and bagged until their use. LD and AA leaves were characterized according to their secondary metabolite composition as indicated by García et al , being tCapra hircus “Creole” breed; 4 mo. old and initial average liveweight: 10.2±0.9 kg) were randomly assigned to three experimental diets  was processed 24 h after slaughter to evaluate quality parameters and the other one (Longissimus lumborum portion) was vacuum-packed and aged for additional two days at 2°C±1°C and, subsequently, processed to evaluate oxidative stability.The feeding trial was conducted at the Experimental Station INTA  in Santiago del Estero, Argentina. Animal handling and experimental procedures were conducted in accordance with regulation procedures for animal welfare of the National Service of Animal Health  of Argentina. Animals were slaughtered at 180 d old at an experimental abattoir. Goats were deprived of food 24 h before slaughter; although, they had free access to water. Slaughter live weight (SLW) was determined immediately before slaughter. Kid goats were slaughtered by exsanguination. The dressed carcass comprised the body after removing skin, head , fore feet , hind feet  and viscera. Tail, diaphragm, kidneys, and fat around the kidneys and pelvic area were kept in the carcass. Hot carcass weight was recorded immediately after slaughter while cold carcass weight (CCW) was measured after 24 h, at 4°C. Dressing percentage was calculated as the percental relation between CCW and SLW (SLW = CCW/LW×100). Objective carcass conformation was measured according to the methodologies recommended by Bonvillani et al , taking 2+ b*2)1/2 and hue angle (H*) as tan−1(b*/a*)×(180/π). Water holding capacity (WHC) was determined by duplicate according to the compression method described by Irie et al [Meat pH and temperature were measured at 24 h post-slaughter between the 4th and 5th lumbar vertebra with a portable pH meter , calibration was performed using buffer pH 4 and 7. Instrumental color  was measured as reported in Luciano et al , using Mie et al .Longissimus lumborum left portion were processed to measure TPC and antioxidant activity. Extracts were prepared homogenizing 5 g of meat in 10 mL of 0.05 M phosphate buffer (pH 7). Mixture was centrifuged at 18,000×g for 20 min at 4°C.Meat samples from The TPC was determined by Folin-Ciocalteu method as suggested by Singleton and Rossi  as folloThe oxidative stability in meat samples was performed mea suring 2-thiobarbituric acid reactive substances (TBARS) levels after different storage conditions.Longissimus lumborum right portion were divided into three fractions. One of them was stored at 26°C for six hours, and the remaining two samples were both vacuum packaged, being the second one stored at −18°C for 30 d and the third one at 4°C, respectively, for 0, 2, 4, and 6 d. Each sample was placed in an individual plastic bag.After the ageing process (3 d), meat samples from the g for 15 min at 4°C. An aliquot of 1 mL of supernatant was added to 2 mL de 0.6% (w/v) aqueous thiobarbituric acid solution into screw cap glass tubes. Tubes were incubated in water bath at 70°C for 30 min, and absorbance was read at 532 nm. The assay was calibrated with solution of known concentration of 1,1,3,3-tetraethoxypropene in distilled water. Results were expressed such as mg of malondialdehyde (MDA)/g of meat.Lipid oxidation was assessed according to the method described by Botsoglou et al . Meat saDetermination of fatty acids profile after derivatization as methyl esters was performed by gas chromatography with flame ionization detector (GC-FID). Intramuscular lipid from muscle sample was quantitatively extracted, according to the method of Bligh and Dyer . The extThe FAME constituents in the sample were identified by comparing the retention times of sample peaks with those of standards obtained from Supelco . Analytic results were expressed as percentages of total fatty acids.All data were analyzed as a completely randomized design with the linear mixed-effects model procedure through the R interface, using INFOSTAT 2014 Statistical Software   for variWhere Yij is the response to diet, μ is the overall mean, Di is the fixed effect of diet, Aj is the random effect of animal and ɛij is the experimental error.Data related to lipid oxidation  were ana lyzed as repeated measures. The model for repeated measures analysis was:Where Yijk is the dependent variable, μ is the global media, Di is fixed effect of diet i, Aj is random effect of animal k, tk fixed effect of time k, (Di×tk) is fixed effect of the interaction between treatment j and time k, and ɛijk is experimental error. Multiple comparisons among means was done using LSD Fisher test (p<0.05).There were no significant differences in average daily gain neither on final live weight nor hot carcass percentage among treatments . Min et Carcass measurements and compactness  were notThe final pH value is a relevant parameter due to its effect on shelf life, color and quality of fresh meat. In the present study, ultimate pH did not differ among treatments . SimilarThe visual appearance of fresh meat is based on color and WHC . Along wIn the present study, no differences were found in meat color due to nutritional treatment . The ligPolyphenols are natural antioxidants that have the ability to reduce oxidative damage, exerting a natural protective action on cell against reactive oxygen species, either trapping free radicals or inhibiting their action. For this reason, in recent years, plants and vegetal extracts rich in polyphenolic compounds have been extensively studied as possible sources of natural antioxidants in animal nutrition. Total phenol contents of meat varied from 10.7 to 11.1 mg GAE/g meat without significant difference among treatments. Although, there were not statistical differences, meat of goats supplemented with AA had numerically the highest content of phenolic compounds followed by those fed with LD.In the same trend, our data did not reveal significant vari ations in the meat antioxidant activity due to the supplemented diets of goats .The effect of different diets on TBARS values in refrigerated meat at 4°C for 6 days are shown in Lipid oxidation was affected by the storage conditions, a significative increase in TBARS along storage period was observed in control group with respect to LD and AA groups. The results obtained for the TBARS values were 2.22 and 3.02 mg MDA/kg of meat for goats supplemented with LD and 2.72 and 3.03 mg MDA/kg of meat for those supplemented with AA at the fourth and sixth days of storage, respectively. The largest increase, up to 5.61 mg MDA/kg of meat after six days of storage, was observed in the control group. Only at the second day of storage was no difference in TBARS accumulation observed among experimental groups. Moreover, it is noteworthy that, meat TBARS of LD and AA groups were around the threshold value suggested as a sensorial detection of meat rancid flavors proposed by Campo et al , which iOn the other hand, TBARS values of meat stored at 26°C for 6 h are shown in Finally, the TBARS values for goat meat stored at −18°C for 30 d are shown in Moringa oleifera leaves, a plant species with a high polyphenol content cultivated in Asia, improves the oxidative stability of the meat [It has been reported that the incorporation into the goat diet of the meat . Neverththe meat . The samthe meat  showed tthe meat .To our knowledge, this is the first report showing the pos itive effect of the incorporation in the diet of goats of woody species LD and AA in oxidative stability of the meat. The protective effect of LD and AA diets against meat lipid peroxidation found in the present work cannot be ascribed to the presence in meat of antioxidant compounds as their contents and antioxidant activity were not significantly different with control. Thus, the observed increase in oxidative stability should be based on the variation of the susceptibility of the meat lipids to oxidation due to a possible change in their composition.cis-10-pentadecenoic acid (C15:1) in muscle AA treatment had significant differences (p<0.05) with the control; being higher in saturated but lower in the unsaturated fatty acids. When woody species were added to the forage, the C18: 2n6c concentration in meat increased by 36% in comparison with goats fed with forage without woody species. The supplementation with woody species did not produce significant differences among SFA levels in contrast to MUFA contents in meat (p = 0.007). The concentration of PUFA was numerically higher in meat of goats supplemented with tannin rich leaves; although, no statistical differences were found.Sixteen fatty acids were identified and quantified in the fat extract of the meat from goats supplemented with LD and AA leaves, which consisted of nine saturated fatty acids , four monounsaturated fatty acids  and three PUFA  . The majThe proportions of PUFA/SFA in the present study were lower than the recommended ratio of 0.45 to balance the negative effects of dietary saturated fat on human health ; being fIn vitro studies showed that the incubation of ruminal fluid with CT reduced the biohydrogenation of linoleic acid [Previous studies have investigated possible feeding strat egies to increase PUFA content in meat and milk, through the manipulation of ruminal biohydrogenation. Min et al  reportedeic acid  and linoeic acid , indicateic acid  studied eic acid , report The higher TPC the lower oxidation level of the meat, suggest ing that these species are an important source of polyphenols, giving caprine meat a greater resistance to oxidation compared to polyphenol free diets. Generally, the present study has demonstrated that LD and AA may be exploited as dietary supplement to improve oxidative stability of meat from goat livestock.Under the experimental conditions of this study, feeding antioxidant-rich native species had no significant effect in terms of animal growth or yield, neither in the meat quality parameters. The most important finding of this experiment was that the inclusion of woody species in the diet of goats promoted not only a lower oxidation status of the meat after slaughter but also increased the resistance of the meat to lipid oxidation under refrigerated or frozen storage. Results also indicated that this enhancement was not due to the increase of the TPC concentration or antioxidant activity in the muscle, but it was probably due to the modulation of the ruminal biohydrogenation producing changes in meat’s lipid profile. The inclusion of AA and LD native species in goat diet enhanced meat oxidative stability.


Internal solitary waves (ISWs) can cause strong vertical and horizontal currents and turbulent mixing in the ocean. These processes affect sediment and pollutant transport, acoustic transmissions and man-made structures in the shallow and deep oceans. Previous studies of the role of ISWs in suspending seafloor sediments and forming marine nepheloid layers were mainly conducted in shallow-water environments. In summer 2017, we observed at least four thick (70–140 m) benthic nepheloid layers (BNLs) at water depths between 956 and 1545 m over continental slopes in the northern South China Sea. We found there was a good correlation between the timing of the ISW packet and variations of the deepwater suspended sediment concentration (SSC). At a depth of 956 m, when the ISW arrived, the near-bottom SSC rapidly increased by two orders of magnitude to 0.62 mg/l at 8 m above the bottom. At two much deeper stations, the ISW-induced horizontal velocity reached 59.6–79.3 cm/s, which was one order of magnitude more than the seafloor contour currents velocity. The SSC, 10 m above the sea floor, rapidly increased to 0.10 mg/l (depth of 1545 m) and 1.25 mg/l (depth of 1252 m). In this study, we found that ISWs could suspend much more sediments on deepwater areas than previously thought. Specifically, we estimated that ISWs could induce and suspend 787 Mt/yr of sediment from shelf to deep-sea areas of the northern South China Sea. The total amount of sediment resuspended by shoaling ISWs was 2.7 times that of river-derived sediment reaching the northern South China Sea. This accounted for 6.1% of the global river-discharged sediment (16.4% of that from Asian rivers) transported to the sea. The interactions between ISWs and the seabed are considered important factors that promote sediment resuspension and transport, as well as seabed deformation and instability5. Notably, these interactions have led to sand waves and scour channels along the shelf break of the northern South China Sea6.Internal solitary waves (ISWs), or internal solitons, are formed in stratified oceans and create remarkable interactions between ocean currents and the seafloor in a relatively short time (tens of minutes)10, and large-scale suspension can erode seafloor sediments and form marine nepheloid layers13. Moreover, ISWs can even shape the slope morphology and affect the sedimentary landscape of the seabed16. Sediment resuspension events triggered by ISWs have been observed and studied in marginal seas, shelf and subaqueous delta areas, such as the Middle Atlantic Bight17, the western Portuguese mid-shelf18, Otsuchi Bay12, the northern South China Sea19, the Oregon shelf 20, Massachusetts Bay21, the California shelf 22, and New York Bay23. The benthic nepheloid layers (BNLs) and intermediate nepheloid layer (INLs) formed by shoaling ISWs have been widely observed in the field18. Previous studies of ISW-induced sediments resuspension have mainly concentrated on shallow sea areas with water depths of less than 200 m. The only deep-sea study ever reported was conducted in the continental shelf break area of the northern South China Sea6. Reeder et al. (2011) observed that ISWs disturbed the seabed sediments and formed 200-m-thick BNLs. However, the results lacked near-bottom flow evidence and have been interpreted in multiple ways5. Additionally, the amount of suspended sediment has never been directly measured nor convincingly estimated.ISWs can cause sediment resuspension and transport in the shoaling process24. Here, ubiquitous ISWs are believed to generate in the Luzon Strait and propagate in the WNW direction, eventually dissipating in the shallow coastal sea, spreading more than 600 kilometres25. Existing studies have concentrated on the Dongsha sea24, and few field attempts have been made to describe seabed sediment resuspension on other foot prints of ISWs. However, with seismic oceanography methods, recent studies have shown that ISWs can reach and shape the seafloor to depths of 400 m or more27, causing potentially substantial sediment resuspension near the seafloor. To address these questions, we studied ISW-induced sediment resuspension based on in situ observations and investigations of marine nepheloid layers. We observed at least four thick (70–140 m) BNLs at water depths of approximately 956–1545 m. Furthermore, we found a good correlation between the timing of the ISW packet and the variations in the deepwater suspended sediment concentration (SSC).ISWs impact the ocean at all depths, but deeper areas have not been studied in detail. The ISWs in the South China Sea are the largest amplitude waves in the world; these waves occur in the tidal period, up to twice a day, and generally in the form of wave packetsA deepwater mooring system T1) was deployed at a depth of 956 m  in the northern South China Sea for nearly 8 consecutive days from June 19th to June 27th, 2017 . Tracklines provided 120–150% seafloor coverage. Using QPS Fledermaus Midwater software (QPS B.V. Netherlands), the water column backscattering data were evaluated parallel and perpendicular to the tracklines to identify water column anomalies.Backscattering data were acquired aboard The profile of water column temperature, salinity, the turbidity and velocity was measured by a conductivity–temperature–depth (CTD) profiling system  assembling with a lowered-ADCP  at T2 , T3  and T4 . The probe extended to 10 m above the seafloor and kept near the seafloor for some time.Selective sediment sampling was implemented nearly the mooring sites to determine the relationship between turbidity (NTU) and the actual SSC (mg/l). Sediments were collected on 142-mm membrane filters (4 µm Nuclepore). Using the least square fitting method, a proportional linear relation between the turbidity recorded by an RBR and the SSC was determined, where SSC (mg/l) = 0.77 × turbidity (NTU). The standard deviation of the error in predicting the SSC was 0.01.Dongfanghong2 ship to investigate the marine nepheloid layer in summer 2017. The route was from Dongsha to Shenhu along the mid-continental slope. The BNLs were produced by the resuspension of seabed sediments29 and BNLs formed by shoaling ISWs have been observed in the continental shelf break of Dongsha6. These particles in the BNL have a lifetime of days, and allow for appreciable lateral advection29. During our observation period, one BNL formed by ISWs was identified and named “A”  and ISW-induced sediment resuspension and the BNL thickness at T1   Fig. . During ′E) Fig. . The dat′E) Fig. , and the30. Therefore, the suspended sediment on the slope moved downward, resulting in a continuously high SSC  and at T4  using the CTD assembling with a lowered-ADCP  than in Shenhu (approximately 0.01 mg/l)  found that when ISWs passed Dongsha, the mixing rate considerably increased32. Additionally, mixing was instantaneously enhanced, and several peaks were observed at different depths when the ISWs was about to arrive or had just passed32. The SSC profile indicated that mixing in the water column caused by ISWs was more significant in Dongsha than in Shenhu.The SSC profile indicated that there were weak INLs above the BNL at T1 and T2 in Dongsha Figs  and 6. H35. Notably, the near-bottom velocity increased as the trough of the ISW approached in our field observation. Based on observations and analyses of sediment suspension events, it is generally believed that the near-bottom velocity is greater than 0.5 m/s, which could resuspend sediments6. Additionally, the velocity observed in this study was enough to suspend the seabed sediment. A near-bottom velocity of a similar order of magnitude was observed in the South China Sea36. Lien et al. (2014) even found that the near-bottom velocity exceeded 1.0 m/s as the trough of the ISW passed a continental shelf break9. The shoaling ISW induced a drastic near-bed current on the continental slope, which resulted in significant resuspension on the seafloor.The mean horizontal velocity (at 12 mab) in the E-W and N-S directions measured by the lowered-ADCP oscillated around the baseline value of ~20 cm/s, and peak values reached 79.3 cm/s Fig. . ISWs am11 m2  transported to the sea and nearly three times the flux of river-derived sediment reaching the northern South China Sea.Recent studies have shown that the global flux of modern river-derived sediment reaching the coasts and oceans under Anthropocene conditions is approximately 12.8 × 10n41 Fig. . The flu30. The BNLs have a lifetime of days, allowing for appreciable lateral advection43. Lateral transport of BNLs can reach especially far and may enter the deep ocean from shallow areas43. INL was constituted by fine particles, and these fine particles in the INL had very low settling velocity (e.g. less than ~5*10−6 m/s), which helped INL exist longer lifetime29. BNLs and INLs are thought to be transport channels both for land-sourced material to the seafloor and for suspended particles from the ocean margin to the ocean interior35. Due to sediment suspended by shoaling ISWs from continental slope to shelf, we inferred that the major suspended sediment ultimately was transported to the basin of the South China Sea.Previous work has discovered that ISWs may erode, resuspend and transport sediments by BNL and subsequently offshore through the generation of INLsThe amount of resuspended sediment in the seabed cannot be ignored in the global oceans. This analysis indicates that ISWs play a controlling role in deep-sea, sedimentary, dynamic processes and seafloor evolution. ISWs can erode the sediment surface and shape continental slopes and shelves, and finally transport suspended particles from the ocean margin to the ocean interior by forming BNLs or INLs.This study found that sediment resuspension by ISWs formed a BNL and enhanced velocity along a continental slope based on mooring system observations and measurements with an lowered-ADCP equipped on CTD. The results provided fundamental evidence that ISWs could suspend sediments in their footprints as they propagated from the continental slope to the shelf of the northern South China Sea. We estimated that ISWs could suspend 787 Mt/yr sediment from the shelf to the deep sea in the northern South China Sea, which accounted for 6.1% (16.4% of the contribution of Asian rivers) of the global river-discharged sediment transported to the sea. The total amount of sediment resuspended by shoaling ISWs was nearly three times that of river-derived sediment reaching the northern South China Sea.There are very few studies that put emphasis on the role of ISWs in deep-sea sediment resuspension, sedimentary and dynamic processes. The results provide an extremely useful case study for similar sediment resuspension events. BNLs and INLs produced by the suspended sediments are ubiquitous in oceans worldwide. Our findings represent a quantitative study on the role of ISWs for sediment transport in the marginal sea that may lead to a better understanding of sedimentary processes and seafloor evolution over geologic time, which is critical for geohazard assessment. Although the South China Sea is morphologically peculiar, we believe that ISW-induced sediment resuspension and transport would occur worldwide and that they have not yet interpreted in this way. This mechanism could be extended to similar areas developed in other marginal seas, especially where ISW activities are frequent and sediment resuspension is violent. These findings reinforce the theory that the relationship between ISWs and sediment resuspension may have been underestimated.supplementary


The objective of this study is to assess and compare the effect of applying a computerised cognitive training programme and virtual environment rehabilitation system on cognitive functions in patients after a stroke. Methods. A controlled trial included 121 persons referred to second stage rehabilitation. The subjects were differentiated into three impact groups by a single blinded trial. Results. The trial revealed that cognitive functions improved in all patient groups (p<0.001). A paired comparison analysis of all groups demonstrated a tendency for cognitive functions, evaluated by the MoCA–LT test, to be more strongly improved in patients who practised a computerised cognitive training programme during their OT sessions than those who did not (p=0.054). Conclusions. The final outcome of the trial was that cognitive functions significantly improved in patients who practised computerised cognitive training programmes or virtual environment rehabilitation systems, compared to those participants who only had occupational therapy sessions. CognModern technologies for cognitive rehabilitation of stroke patients are becoming more accurate and effective . TechnolScientific papers have suggested that CCT may improve patients’ cognitive functions, especially working memory and motivation . ComputeOther researchers have noted that conventional rehabilitation exercises may seem tedious due to their repetitive nature . MoreoveStudy objective. To assess and compare the effect of applying a computerised cognitive training programme and virtual environment rehabilitation system on cognitive functions in persons with ischaemic stroke.22.1A randomised single blinded trial was performed in two Kaunas city hospitals after obtaining Kaunas Regional Biomedical Research Ethics Committee permission (No. BE–2–33). This study describes the outcome of the trial that was conducted in Lithuania and lasted for three years. The study objectives were introduced to trial participants and they voluntarily agreed to sign an informed consent form to participate. The inclusion criteria were as follows: subjects with first-time ischaemic stroke (10–14 days after the stroke), Barthelindex (BI) 50–65 points, Mini-Mental State Examination (MMSE) ≥11 points, subject’s agreement to participate in a trial, and vision and hearing suitable to evaluate cognitive functions and apply impact measures. Exclusion criteria involved BI from <50 or >65 points, MMSE <11 points, repeated stroke, subjects unable to speak or have diagnosed aphasia, neglect syndrome, and other neurological or mental health disorders. These criteria were chosen according to the law of the Ministry of Health of the Republic of Lithuania, and patients were treated in the Department of Physical Medicine and Rehabilitation for 32 days when they showed a BI of 50–65 points and results of the MMSE of 11–30 points. The trial involved 126 subjects with ischaemic stroke who were referred to rehabilitation. Patients were randomly assigned to study groups with a 1: 1: 1 allocation ratio, according to the rehabilitation registration journal. The first trial group (T1) consisted of 42 subjects, the second trial group (T2) involved 42 subjects, and the third trial group (T3) also consisted of 42 subjects. A total of 121 subjects established the trial, and 5 subjects left the trial because of the following reasons: 2 subjects died, 2 subjects discontinued the trial due to the impairment of their health condition, and one subject refused to participate in the trial .Before and after the trial, cognitive functions of the subjects were evaluated with the MMSE, Montreal Cognitive Assessment test  that was validated and adapted in Lithuania (MoCA–LT).2.2The T1 group participated in a programme that included individual occupational therapy (OT) sessions (five times a week) with an aim of improving the existing cognitive impairments. During the sessions, participants were given tasks to train spatial perception, memory, attention concentration, and problem solving. All these functions were trained with the help of conventional “pencil–and–paper” cognition training tasks.PssCogRehab 2012, USA) three time a week. Individual OT included conventional “pencil–and–paper” cognition training tasks. Computer–based programmes that were practised three times a week included tasks from four modules , and they were performed by a subject sitting in front of the computer screen. The tasks of these modules trained the subjects’ memory, problem solving, attention concentration, and spatial perception. The difficulty of the task was selected according to the subject’s cognitive function state.The T2 group participated in a programme that aimed to improve the existing cognitive impairments; this involved individual OT two times a week and individual OT sessions with a computerised cognitive training programme  three times per week. This system comprises a Kinect camera and specialised games for rehabilitation. Patients involved in VE rehabilitation sessions practised different game programmes and performed these programmes with hand movements sitting or standing 2–3 metres away from a motion sensor or camera. The following modules were applied: Ball, React, Cleaner, Space, Warm up, Maze, Sorter, and Gym. Programmes were selected depending on the participant’s cognitive function disorders.The T3 group participated in a programme that provided individual OT two times a week, which was the same as the T1 group, and OT sessions included practised VE rehabilitation system activities , minimum value (xmin), maximum value (xmax), and arithmetic mean Statistical data analysis was performed with IBM3The study presents the results of the 121-subject trial, of which there were 75 (61.98 %) females and 46 (38.02 %) males. The average age of the subjects was 72.61 years , the average age of the males was 70.17 , and the average age of the females was 74.08 . Before the trial, subjects were differentiated into homogenous groups according to gender, education, age, side of damage, and marital status ; there w2(2)=8.916; p<0.05). Paired comparison analysis of all groups revealed significant result differences between the T1 and T2 groups (p=0.026), and between the T1 and T3 groups (p=0.033). Overall, it was identified that cognitive functions were significantly improved in participants who had OT sessions involving the CCT programme or VE rehabilitation system.The study findings  indicate2(2)=5.139; p=0.077). However, after the trial, MoCA–LT test findings showed differences between all three trial groups (χ2(2)=6.555; p<0.05). Paired comparison analysis of all groups showed a tendency towards the improvement of cognitive functions being stronger in patients who practised the CCT programme during the OT sessions (p=0.054).The analysis of the MoCA–LT test results  revealed2(2)=7.834; p<0.05). Paired comparison analysis of all groups showed a significant difference only between the T1 and T2 groups (p=0.038). Analysis of the cognitive function changes reflected in the MoCA–LT test results revealed that, on average, cognitive functions in the T1 group improved by 3.10±3.60 points. In the T2 group, it improved by 5.90±2.53 points, and in the T3 group, it improved by 3.33±3.01 points. The analysis of the changes in all groups reflected in the MoCA–LT data, demonstrated that the results were different in all three trial groups (χ2(2)=20.142; p<0.001). Paired comparison analysis of all groups showed a significant difference in cognitive function changes between the T1 and T2 groups (p<0.001).The comparison  of the a4The choices of science–based interventions related to the rehabilitation of cognitive functions after stroke are increasing. However, reviews in the scientific literature of trials combining the impact of two cognitive function training programmes (computer–based cognitive training and VE rehabilitation systems) are infrequent during the early stage of rehabilitation after stroke.CoTras and identified that the CCT programme greatly improved patients’ cognitive functions during the early stage of rehabilitation after stroke [The results of this study revealed benefits of CCT programmes and VE systems in the process of rehabilitation. Park J.H. et al. 2015) practised a computerised programme 15 practir stroke . The abor stroke , 23, 24.r stroke . Neverthr stroke .To identify the state of the cognitive functions, the studies employed numerous validated tools, such as MMSE, Neuropsychological Test Batteries, MoCA, Loewenstein Occupational Therapy Cognitive Assessment, and others . The preRecommendations for cognitive rehabilitation are rather general and usually differ at a low evidence–based level . Most stA large number of trials apply cognition training interventions at a later stage after stroke treatment; however, as some experts have stated, the recovery of cognitive functions is faster when the interactive rehabilitation programmes are practised during the acute or subacute stroke stage , 20, 21.As the scientific literature suggests, there is a wide variety of CCT programmes and VE rehabilitation systems aimed at training cognitive functions and offering different levels of duration and intensity , 12. In American Stroke Association recommendations state the expedience of an enriched environment to increase the subject’s engagement in cognitive activities (evidence level A) . TherefoOther researchers have stated that CCT programmes as well as VE rehabilitation systems might be very beneficial if practised in combination , 29, 36 5The generalisation of the study results highlight several limitations. First, we would recommend the analysis of a general change in cognitive functions in a detailed evaluation of particular cognitive functions. In addition, we would recommend successive monitoring of the impact of the practised CCT programmes and VE rehabilitation system for the assessment of a persistent benefit. At the beginning of the trial, the elderly intended to refuse to participate and indicated that they did not know how to operate the interactive means, and they were stressed because of lack of self–confidence due to a possible inability to perform the given tasks properly. Another limitation of this study was that we had to overestimate the limits of self-sufficiency (BI 50-65) and cannot say what the adaptability of this data would be in another patient’s self-sufficiency level. Therefore, it would be appropriate to design a simpler operation for the applied technologies.6Cognitive functions after rehabilitation have improved for all groups. Better estimates of cognitive functions were a computerised cognitive training programme or virtual environment rehabilitation system practised during OT sessions significantly improved the cognitive functions of patients with ischaemic stroke than in a group of people who were only in occupational therapy sessions. Applying innovative rehabilitation measures during OT sessions can assist patients in achieving better outcomes as well as restoring, stabilising, and improving cognitive functions during the early stage of rehabilitation after an ischaemic stroke.