This manuscript is a survey of the halachic attitudes toward organ transplant procedures from a living donor which can be defined as life-saving procedures for the recipient or at least life-prolonging procedures. Three fundamental problems concerning the halachic aspects of such transplantation are discussed in detail: the danger to the donor, donation under coercion, and the sale of organs and tissues. The terms “halacha” and “Jewish law” are defined in the introduction. The halachic discussion of ethical dilemmas in medicine is ancient, its principles interspersed throughout the Bible and the Talmud. Over all historical periods halachic literature has included discussions on medical topics.halachic aspects of transplantation from living donors, the terms halacha and Jewish law must be defined and understood.Before any discussion on the Halacha includes those issues of halachic rights and obligations as well as issues of religion and morality – in other words, those commandments that deal with interpersonal relationships as well as with the relationship between man and God. This category is broader than “Jewish law”.Jewish law is a modern concept that includes those issues in halacha that are the subjects of legal rights and obligations. Such subjects are considered legal issues under other legal systems, as opposed to religious and moral issues which are not. If a somewhat simplified definition will suffice, then we may say that Jewish law deals principally with interpersonal laws and almost not at all with the laws between man and God. The obligations of the physician, the patient, and society, for example, belong in Jewish law, but the laws on practicing medicine on Shabbat or on Yom Kippur do not. They belong, however, in the broader field of halacha.There are three fundamental problems concerning the halachic aspects of transplantation from living donors: 1) The danger to the donor; 2) donation under coercion; and 3) sale of organs and tissues.Pikkuach Nefesh. It should be emphasized that where a procedure is not a life-saving measure, all halachic authorities agree that one may not significantly endanger a donor’s life.We shall discuss here only transplants which can be defined as life-saving procedures for the recipient or life-prolonging procedures. In halachic terms these are cases of 1)Everyone is obligated to try to save his own life or the life of another who is in danger. There are two aspects to this commandment: the preservation of life and the restoration of “lost property”. The commandment of the preservation of life is derived from the verse: “You shall therefore keep my statutes and my judgments: which if a man do, he shall live by them” (Leviticus 18:5). From this the sages deduced: You shall live by them, but not die by them.The preservation of life overrides all but three prohibitions of the Torah: idolatry, illicit sexual intercourse, and the shedding of blood.5Suicide is forbidden as part of the prohibition of killing.Halacha is clear in two cases. If Mr A’s life is in danger and Mr B can save Mr A without endangering his own life, he must do so. If Mr B can only save Mr A by sacrificing his own life, he may not do so May a patient attempt to save his own life by compelling another to donate an organ? Consider, for example, a patient with a rare blood type who is injured and whose life depends on an immediate blood transfusion. The blood bank does not have the critical blood type available but knows of a suitable donor. That potential donor refuses to donate blood, even though this means that the patient will die. May the potential donor be compelled to give blood?Related questions arise when the potential donor is legally incompetent to give consent to the procedure. A retarded or autistic donor, or a donor who has not yet reached the age of majority, is legally incompetent to consent to any procedure, and the guardian’s consent is valid only when it is in the interests of the ward. May such an incompetent person be accepted as a donor of blood in order to save the life of the injured patient?There are two halachic prohibitions in drawing blood from a donor without his consent: 1) The prohibition of “theft”; and 2) the prohibition of injuring a person.The prohibition of “theft” is derived from the verse: “Thou shalt not steal”,The preservation of life overrides all but three prohibitions of the Torah . One might thus conclude that the prohibition of theft and injury to others are suspended in life-saving situations.Accordingly, it might appear that one may save one’s life by compelling a suitable donor to give blood, just as one may save one’s life by eating on the Day of Atonement, by setting aside the Sabbath laws, or by eating otherwise prohibited food.Nevertheless, in cases of theft or injury to another, a second party is involved, and the circumstances are therefore not comparable. Discussions of similar point appear in the Talmud, the works of early and more recent authorities.61abizraihu.Most authorities hold that the saving of life does not supersede any “act related to killing” – Rabbenu YonahDoes the preservation of life supersede all but three prohibitions?Is personal injury an act “related to killing?”If so, does the preservation of life supersede acts related to killing?The question of coercing a donor to donate an organ or body tissue in order to save the life of another is not simple. Its solution depends on a number of fundamental factors:may not attempt to save his own life by compelling another to donate an organ.These three questions are the subject of ongoing controversy among halachic authorities. Nevertheless, a patient 3)The issue of sale of organs and tissues is a sensitive one; the emotional aspects of the issue cannot be neglected in the discussion. Nevertheless, the halachic aspects of the issue must be discussed dispassionately in the light of authoritative sources. Here we shall deal with the halacha pertaining to the sale of human hair, blood, and kidneys.The MishnahOne might say that there is no essential difference between the sale of hair intended for a wig and skin intended for grafting onto the head of another. But one might distinguish between the procedure of cutting the hair, which is permitted, and the procedure of removal of a donor’s skin, which might be considered to be injury and thus prohibited. Moreover, hair regrows as contrasted with organ or tissues. This brings us to the basic question of a person’s right to injure himself.own his body. According to Rabbi Shlomo Yosef Zevin a person does not own his body.All authorities agree that it is prohibited to injure oneself irreversibly.This is derived from the principle that wanton destruction is not permissible.78There are differences of opinion among the sages in cases where one “injures” oneself for beneficial effects. According to one Talmudic source87In view of this, the utilization of organs and body tissues for purely commercial purposes is not permissible. Similarly, it is prohibited to donate a kidney for research or industrial purposes if the benefit to the donor is purely financial. On the other hand, cutting the hair involves no injury, and it is therefore permissible to use hair for purely commercial reasons.Blood donations fall somewhere between the examples discussed above. In drawing blood there is only minor discomfort. Is this similar to cutting hair, which is not considered an injury, and therefore permitted? Or is drawing blood more like kidney donations? Rabbi M. Feinstein tended to permit drawing blood for purely commercial reasons.88Although one may not remove a kidney for mere financial benefit, one may surely remove it to transplant it for the prolongation of life. Even relief from suffering or improvement of the quality of life is considered to be of great enough benefit to justify the injury involved in removing a kidney.As a rule, one should not accept payment to fulfill a commandment of the Torah.Society may legislate to prevent the exploitation of its poorer members.Informed consent and a firm decision to sell are necessary prerequisites for removal of an organ or tissue, and for transfer of ownership to the purchaser.When there is no prohibition of injury to the donor of an organ or tissue, does the donor have a right to demand payment? In principle, it would seem that the donor should have the same right to sell a kidney or blood as he has to sell his hair. But three points might restrict this right:In principle one may not insist on monetary compensation for teaching Torah.91This principle is not limited to instruction in Torah. It encompasses the fulfillment of all commandments.Although a healer may not demand compensation for his efforts in healing, he may request compensation for his expenses, his time, and any medications or devices which he gives the patient.“Tsa’ar” in the terms of the Mishnah. Therefore, a donor has every right to demand compensation for a donated organ and for the suffering incurred by its removal, even when such an act is considered as a great mitzvah.It is obvious that the loss of an organ can, to some extent, be evaluated in terms of money. It can be concluded also from the Mishnah.any medical procedure of a life-saving nature.A reason presented for permitting midwives to receive compensation for the performance of their occupation on the Sabbath is: “because if they knew that they would not be paid, they might not come”.Even if physicians were not allowed to receive compensation, there is a fundamental difference between the donor of an organ and a physician. A physician is charged with the commandment to heal. He cannot exempt himself from this obligation, and it may be argued that one who should not refuse rendering medical service has no claim to compensation.In summary, the general prohibition of compensation for fulfilling a commandment does not conflict with the right of a donor to demand and receive payment for his suffering and for the organs or tissues donated.It would seem that in a cruel world there is real danger of an organ market in which the affluent might purchase an organ from the poor. This is an example of exploitation of the poor by the rich. In order to prevent such legalized exploitation, it would be appropriate to introduce legislation regulating the sale of human organs and tissues . Today, as we have no central halachic authority to legislate universally binding laws, rabbinic bodies have jurisdiction only in those locations which have accepted their authority.102In summary, unless such a prohibition is legislated, we cannot prohibit the sale of organs for purely exploitative reasons, whether the donation of an organ may lengthen human life, or where it may improve the quality of life.Secular Israeli law requires the patient’s signature on a consent form prior to surgery. The law stipulates the formula to be used. A physician is also required to sign a form certifying that he has explained to the patient everything contained in the form, that the patient fully understood, and that the patient signed the form in his presence.The requirement that the patient fully understand the need for and the possible results of the surgery is impracticable in many cases. In fact, this requirement is fulfilled in only a minority of cases. Generally speaking, the patient has neither the medical knowledge nor the ability to weigh the matter seriously. The physician’s signature does not change these facts.From the halachic point of view a surgical procedure which may save the patient’s life does not require his consent. But the removal of an organ to save another patient is different. In such a case consent of the donor is of great significance. Without explicit prior consent the donor might subsequently claim that consent was given in error and that he had never intended to allow removal of an organ or tissue from him.It is doubtful if the profit-seeking donor always properly understands the medical issues involved in the donation. The donor’s need for money may lead him to ignore the medical consequences of his donation. As a result, the donor may be considered as not fully informed, and his consent might thus not be valid.If a human organ is sold under coercion, the sale seems to be invalid since it fails to comply with one of the basic conditions of “meeting of the minds”. A donor who sells an organ because of urgent financial need is in a state of coercion. Payment for the coerced sale does not create a situation of consent unless the seller receives full value and loses nothing on the transaction.104“There is therefore no reason whatever to ban one who donates a part of his body from requesting and receiving payment for this. The amount of payment can be stipulated in advance and agreed between the donor and a member of the family of the recipient of the transplant ... Such payment, provided that it is within reasonable limits, need not be seen as unethical, since the donor undergoes physical and at times mental suffering, and as stated a person does not waive his rights to his organs ...the donor himself is allowed to receive payment for his donation. Any intermediary acting between the donor and the family of the recipient, whether an individual or an organization that undertakes to deal with the matter, must act strictly within the halacha, which regards this is as trouble that everyone is obliged to take, ‘restoring the [safe] body’ to its owner. They are therefore forbidden to accept any payment other than compensation for abandoning other work, as explained above regarding restoring lost property. This should certainly be embodied in statutory law, to save us from the danger of a trade in human organs developing.”105“At the same time, it must be pointed out that only Rabbi Shlomo Zalman Auerbach has a more consistent opinion. He wrote that in order to save life, both the donor as well as the mediator, are allowed to receive compensation.107There is no halachic prohibition against receiving compensation for donated organs.Sale of an organ as a result of desperate financial distress may create a situation of coercion without full value being paid. Such a situation lacks “complete consent”, and the sale might be void.A donor’s incomplete understanding of the medical consequences of the removal of an organ may invalidate the sale.In light of the differences in various cases, the donation of organs for payment should be regulated and requires fully informed prior consent. This should eliminate exploitation on account of uninformed consent.


There is increasing interest by chiropractors in North America regarding integration into mainstream healthcare; however, there is limited information about attitudes towards the profession among conventional healthcare providers, including orthopaedic surgeons.We administered a 43-item cross-sectional survey to 1000 Canadian and American orthopaedic surgeons that inquired about demographic variables and their attitudes towards chiropractic. Our survey included an option for respondants to include written comments, and our present analysis is restricted to these comments. Two reviewers, independantly and in duplicate, coded all written comments using thematic analysis.487 surgeons completed the survey (response rate 49%), and 174 provided written comments. Our analysis revealed 8 themes and 24 sub-themes represented in surgeons' comments. Reported themes were: variability amongst chiropractors (n = 55); concerns with chiropractic treatment (n = 54); areas where chiropractic is perceived as effective (n = 43); unethical behavior (n = 43); patient interaction (n = 36); the scientific basis of chiropractic (n = 26); personal experiences with chiropractic (n = 21); and chiropractic training (n = 18). Common sub-themes endorsed by surgeon's were diversity within the chiropractic profession as a barrier to increased interprofessional collaboration, endorsement for chiropractic treatment of musculoskeletal complaints, criticism for treatment of non-musculoskeletal complaints, and concern over whether chiropractic care was evidence-based.Our analysis identified a number of issues that will have to be considered by the chiropractic profession as part of its efforts to further integrate chiropractic into mainstream healthcare. In 2006 the Chiropractic Strategic Planning Conference proposed a set of recommendations for advancing the chiropractic profession in North America . These iWe recently surveyed 1000 North American orthopaedic surgeons (49% response rate) to inquire about their use of, and attitudes towards, chiropractic . ApproxiWe have reported details of our questionnaire development and administration elsewhere . In brierd  survey prior to faxing in an effort to encourage completion of the instrument. The McMaster University Research Ethics Board approved our study.From July 2006 to June 2007 we administered our survey to a random sample of 500 Canadian orthopaedic surgeons, and from July 2007 to June 2008 we administered the same survey to a random sample of 500 American orthopaedic surgeons. We administered surveys by fax, and all surgeons' offices were telephoned prior to sending a survey to confirm their presence and fax number. Participants were provided with a disclosure letter detailing the intent of the survey and explicit instructions that, should they choose not to complete the survey, they could check a box on the cover page indicating that they did not wish to participate and fax it back to our attention. At 4 and 8 weeks following the initial mailing, we re-faxed the questionnaire to all non-responders. We telephoned each office that received a 3a priori, only to present sub-themes that were endorsed by a minimum of 3 survey respondents. Our selection of quotations for presentation was guided by consensus among reviewers that selected statements were particularly informative, representative and succinct.We have reported our analysis of respondent's survey data elsewhere  and the 2 test. We explored for differences in mean CAQ scores between survey respondents who provided written comments and those who did not to explore for the presence of an attitudinal bias. All comparisons were 2-tailed and we set our level of significance at p ≤ 0.01 to account for multiple comparisons. We performed all analyses using PASW Statistics 18.0 .We generated frequencies for demographic characteristics and calculated mean CAQ scores for respondents who provided written comments and checked for differences between Canadian and American surgeons with an independent samples t-test and χSurgeons completed 487 of 1000 surveys, of which 174 (36% of respondents) provided written comments. Characteristics of surgeons that provided written comments are provided in Table Wide range of practice - some are evidence based...others do all sorts of crazy stuff". Eleven respondents acknowledged difficulty in answering our survey, in that their responses to a number of questions would depend on the type of chiropractor under consideration: "Obviously not all [chiropractors] are the same...This questionnaire relates to the majority of [chiropractors], not all". Fourteen respondents dichotomized by 'good' and 'bad' chiropractors, and 10 acknowledged similar limitations in orthopaedics: "There are good chiropractors and bad orthopaedic [surgeons] and vice versa", and: "I explain to my patients that there are excellent chiropractors just like there are bad orthopaedic surgeons. But I wouldn't pick one out of a book or from an ad". Six respondents felt that diversity among practitioners was harmful to the chiropractic profession: "I think that chiropractic is hurt by the fringe treatments that some offer", and: "... some unethical chiropractors have given the profession a bad name".The most commonly endorsed theme was diversity within the chiropractic profession: "I feel chiropractic can be beneficial in helping mechanical low back pain", and: "I find their usefulness is limited to lumbar mechanical back pain care". Sixteen respondents endorsed an expanded role for the treatment of musculoskeletal disorders in general: "I think chiropractic care is beneficial for musculoskeletal pain if there are no risk factors", and: "chiropractors have a role in chronic musculoskeletal pain control". Eight surgeons suggested a broad role in the management of non-surgical injuries: "I have a very good relationship with a [chiropractor]...He has helped many members of our sports teams with hands on care of acute and chronic non surgical injuries".Three respondents felt that chiropractic care for spinal complaints was effective, and 16 endorsed care specifically for low back pain: "No objective long term benefits", "Chiropractors understand that a majority of back or neck strain ailments will resolve with or without 'therapy'", and: "It is only after all the therapy visits that do not require pre-authorization or the insurer notes no improvement that the worker gets referred for an orthopedic evaluation".Fifty-four surgeons noted concerns with different aspects of chiropractic care. Some focused on non-spinal conditions, and 3 with chiropractic management of structural scoliosis; however, most respondents (n = 13) in this category felt that chiropractic care provided short-term benefit only or was ineffective: "I have a huge problem with chiropractors claiming to treat asthma and ear infections among other non-musculoskeletal problems", and: "most chiropractors (in my view) are very helpful for spinal complaints. Manipulation for asthma, high blood pressure, breech presentation is quackery - not evidence based. We all need to stick to what we do best".The second most commonly endorsed concern (n = 12) took issue with treatment of non-musculoskeletal complaints: "Chiropractors are dangerous when they portray themselves as replacements for patients' primary care physicians", and: "There are 2 breeds of chiropractors, one who treats only musculoskeletal problems and knows their limitations and does not try to treat everything with manipulation. Then there is the other group who feel they are family physicians which they are not and try to treat all problems like asthma or infections with manipulation". Eleven respondents expressed concerns with adverse events associated with chiropractic care: "In the last 2 years I have seen 2 patients with vertebral artery dissection within 10 days of chiropractic neck manipulation", "Have on several occasions done emergency surgery resulting from chiropractic management", and: "In the orthopedic department in which I worked we saw paraplegics produced by spinal manipulation by chiropractors".Eleven respondents raised concerns regarding the provision of general medical care by chiropractors: "The concept of 'adjusting subluxations' is nonsensical. 'Adjusting' hips and sacroiliac joints is nonsensical", "Why do chiropractors cling to an antiquated theory as inappropriate as 'the humors'?", "Every patient is told that one leg is longer than the other as the root of their medical problems", and: "When chiropractors change the principal that 'all disease emanates from the spine and can be cured by manipulation' they will be more welcome in the medical community and hospitals. It is no question that they are the best at manipulation".Surgeons were largely critical of chiropractors' interactions with patients. Most concerns were directed towards chiropractic terminology, which respondents felt was often misleading or incorrect: "False information given to patients - I had a cerebral palsy child going to a chiropractor for 5 years to improve his gait!", and: "...much of their 'explanations' are based on grains of truth that are distorted to support their claims"; however, 6 did acknowledge non-specific benefits of the clinical encounter: "Chiropractors are very good at taking care of the 'worried well' patients", and: "Very complimentary (sic) to musculoskeletal care - they often talk to patients for great lengths of time while treating, unlike ourselves".A number of respondents felt that chiropractors often made false or exaggerated claims: "Life chiropractors are scary", and "I visited the Canadian Memorial Chiropractic College in Toronto. I was impressed with the similarity of their basic science curriculum - same books as used in medicine - and the extent of information covering all symptoms. This does not seem to be the case for all schools in North America". Two respondents noted their perception that "younger [chiropractors] are better prepared and trained than their older colleagues", while 2 others reported concern regarding "a lack of diagnostic skills". Two other surgeons noted their experiences with both well and poorly trained chiropractors: "I personally use and refer to several extremely well trained [chiropractors] in my area; however, there are much more poorly trained [chiropractors] than well trained [chiropractors]. Overall, appropriate treatment by well trained [chiropractors] is very valuable".Ten surgeons noted concerns with chiropractic training, with two respondents noting a lack of standardization and 2 mentioning specific schools: "All chiropractic care could be better managed by physiotherapy and occupational therapy", "Manipulative therapy is not the exclusive domain of chiropractors. Many physical therapists and athletic therapists are also skilled with that technique and deserve equal recognition", and: "Manipulation of back for mechanical back pain can be done by physiotherapists. There is no need for chiropractors".Eight surgeons noted their perception that the helpful aspects of chiropractic care could largely be provided by other therapists: "Major problem as I see it, chiropractors tend to place patients on an 'x amount of treatment' plan such as 20 visits. In my opinion they need to perform the service and see if the patient responds and proceed from there. Not just say 'you need an x amount of treatment' plan", "The concept of repetitive on-going care (maintenance/prevention) is completely contrary to common sense and science. This is what I object to", and: "Many chiropractors take advantage with the lawyers on auto accident cases. Over treating until the medical payments are exhausted". Fifteen surgeons also noted concerns that some chiropractors may be overly financially motivated: "I believe that most chiropractors are ethical...The trouble is that there are many who team up with unethical legal/rehab facilities and abuse the system for personal gain, partly at the detriment of patient care", and: "only driving force behind many chiropractor's treatment plansMoney is the ".Concerns over excessive treatment by chiropractors were raised by 18 respondents: "Too many chiropractors engage in unethical practices", and "When I started practicing 28 years ago several chiropractors in the area would refer me patients and I'd refer them back. That pattern ended 15 years ago for unknown reasons except many chiropractors in this area are now employees of entities that have sales quotas, profit goals, and widening 'healthcare' ambitions that seemingly come first to patient care". Five respondents advised that the chiropractic profession requires stronger oversight: "Their profession needs more careful government scrutinizing and control", "I think that most chiropractors are honest...unfortunately they are overshadowed by dishonest ones...my opinion is that chiropractors should establish an honest ethics committee that [medical doctors can report] despicable behavior", and: "immenselyMore self policing by chiropractors at the state level would help them ".Five surgeons noted more general concerns with unethical behavior: "Most students now have undergraduate degrees and are relatively well educated in basic sciences in their first 2 years . The problem begins when they move into their clinical years where education becomes 'indoctrination'. Extracts from basic science are selectively constructed into a theory of practice with little if any real scientific basis", "Chiropractic care would be better accepted in medical circles if it were evidence based/scientific and realistic in its' claims and applications to patients", and: "There is no place for chiropractors to work side by side with scientific medical practitioners". Six surgeons advocated for more research into chiropractic treatment: "We need some evidence based chiropractic. Publications in orthopedic journals or medical journals with peer review", and: "[Chiropractors] need to produce more clinical studies of effectiveness".Twenty respondents took issue with the scientific basis underlying chiropractic: "Chiropractic has been a valuable accessory to my practice. In particular dealing with international, national and professional athletes", "I have a very good relationship with a [chiropractor]...given a diagnosis is established, his care is very efficacious!", and: "I go to one myself for neck and back problems". However, four surgeons noted negative experiences: "I have personally cared for at least 2 patients that had wrist tendonitis that were off work for ≥1 year [attending chiropractic care] that only needed 1 shot to be cured", and another 4 expressed uncertainty as to the role of chiropractic in healthcare: "I need more knowledge to utilize chiropractors better!", and: "Unclear at this point what their role is".Thirteen respondents reported positive personal experiences with chiropractic: "We found that written comments by orthopaedic surgeons most frequently endorsed diversity within the chiropractic profession as a barrier to increased interprofessional collaboration. Surgeons identified that training, diagnostic ability and treatment skills were highly variable within the chiropractic profession, and highlighted that unethical behaviors by some affected perceptions of the profession as a whole. Respondents largely felt that chiropractors provided helpful care for musculoskeletal complaints, and particularly mechanical low back pain, but were concerned about the potential for adverse events and were very critical of chiropractic management of non-musculoskeletal conditions. Respondents called for greater integration of evidence-based practices within chiropractic, more chiropractic-related research, and increased oversight by regulatory bodies.Strengths of our study include a detailed assessment of all written comments, conducted independently and in duplicate and guided by a comprehensive coding strategy; however, our study does have limitations. Surgeons who provided written comments tended to have more negative attitudes towards chiropractic. Furthermore, we coded all unique themes or sub-themes from each survey which means that some respondents contributed more content to our analysis than others. Generalizability of our findings should be explored in a representative sample of North American orthopaedic surgeons.Respondents' general perceptions that chiropractic treatment, primarily defined by joint manipulation, has been found to be effective for some musculoskeletal complaints , but not for non-musculoskeletal conditions, is generally consistent with current evidence -14. DespUnethical practices by some chiropractors have also been acknowledged by the profession. In 2008 the Ontario Chiropractic Association conducted a membership survey  which found the majority (67%) "felt that the practices of their peers are in some way negatively influencing perceptions of the profession" . OpinionOur findings suggest that there are a number of concerns held by North American orthopaedic surgeons that should be addressed as part of any efforts to facilitate greater integration of chiropractic into mainstream healthcare. Our analysis of written comments provided by surgeons emphasized the importance of a number of items that we had incorporated into our survey. These include chiropractic's role in managing musculoskeletal and non-musculoskeletal complaints, perceptions of excessive treatment by chiropractors, and whether or not chiropractic is perceived as evidence-based. New items suggested by our findings were diversity within the chiropractic profession as a barrier to interprofessional collaboration, perceptions of chiropractors as primary care providers, questions regarding where chiropractic care may be integrated into mainstream healthcare, and general concerns regarding adverse events associated with chiropractic care.Our analysis of written comments provided by North American orthopaedic surgeons who responded to our survey identified a number of issues that will have to be considered by the chiropractic profession as part of its efforts to further integrate chiropractic into mainstream healthcare. These include: diversity within the chiropractic profession as a barrier to interprofessional collaboration, chiropractic's role in managing musculoskeletal and non-musculoskeletal complaints, perceptions of excessive treatment by chiropractors, whether or not chiropractic is evidence-based, whether or not chiropractors are primary care providers, where chiropractic care may be integrated into mainstream healthcare, and general concerns regarding adverse events associated with chiropractic care.The authors declare that they have no competing interests.JWB conceived of and coordinated the study, performed the statistical analysis, and drafted the initial manuscript. CJ, TN, RR and DT administered the surveys and telephoned surgeons' offices. JWB and JJ coded all data. All authors contributed to survey design, and read and approved the final manuscript.


The spectrum of cancers seen in a hospital based Lynch syndrome registry was examined to compare the incidence of colorectal versus extracolonic cancers by sex, race, mismatch repair (MMR) gene mutation, age at diagnosis and index cancer. We also examined rare extracolonic cancers that are not considered to be part of Lynch syndrome.There were a total of 463 cancers recorded in 239 patients from 150 families enrolled in the Lynch syndrome registry at MD Anderson Cancer Center. Each of these patients had a confirmed germline MMR mutation. Overall there were 218 (47.1%) colorectal and 245 (52.9%) extracolonic cancers. There was no difference in the total number of colorectal and extracolonic cancers in MLH1 mutation carriers (50.3% vs 49.7%) whereas for MSH2 and MLH6 mutation carriers there were more extracolonic than colorectal cancers overall (54.2% vs. 45.8% for MSH2 and 61.9% vs. 38.1% for MSH6). Men had fewer extracolonic cancers than colorectal (44.6% vs. 55.4%), whereas women had more extracolonic cancers than colorectal (58.9% vs. 41.1%). The mean age at diagnosis for extracolonic cancers was older than for colorectal, 48.6 vs. 44.5 years (P=<0.001). There were no significant differences by race between the colorectal and extracolonic cancers in Whites, African Americans and Hispanics.The index cancer was colorectal in 54.8% of patients. Among the extracolonic index cancers endometrial/uterine was the most common (29.6%) followed by skin . Other index cancers were renal/ureter/bladder (10.2%), upper GI/small intestine (6.5%), ovary (5.6%) and hepatobiliary/pancreas 3.7%). It was interesting to note that the index tumor was breast cancer in 5 patients (4.6%) although breast cancer is not considered part of Lynch syndrome [.7%. It wIn this study, we found that Lynch syndrome patients can commonly present with cancers other than colorectal and endometrial. This has clinical relevance both for diagnosis of Lynch syndrome and surveillance for cancers of different sites during followup of these patients.


Drosophila melanogaster embryos, as follows. 1) We confirmed that HR outcomes are enhanced relative to the alternative nonhomologous end joining (NHEJ) repair pathway in flies lacking DNA ligase IV. 2) The minimum amount of homology needed to support efficient HR in fly embryos is between 200 and 500 bp. 3) Conversion tracts are very broad in this system: donor sequences more than 3 kb from the ZFN-induced break are found in the HR products at approximately 50% of the frequency of a marker at the break. 4) Deletions carried by the donor DNA are readily incorporated at the target. 5) While linear double-stranded DNAs are not effective as donors, single-stranded oligonucleotides are. These observations should enable better experimental design for gene targeting in Drosophila and help guide similar efforts in other systems.Gene targeting is the term commonly applied to experimental gene replacement by homologous recombination (HR). This process is substantially stimulated by a double-strand break (DSB) in the genomic target. Zinc-finger nucleases (ZFNs) are targetable cleavage reagents that provide an effective means of introducing such a break in conjunction with delivery of a homologous donor DNA. In this study we explored several parameters of donor DNA structure during ZFN-mediated gene targeting in  Zinc-finger nucleases (ZFNs) , their cDrosophila greatly improves the recovery of HR products  and nonhomologous end joining (NHEJ) . When th1118w stock were used as wild types. Two lig4 stocks carried the 169lig4 mutation, either in a wild-type or 1118w background (EP385lig4 mutation (Bloomington Stock Center). Stocks and crosses to reveal new rosy (ry) mutations were as described previously  and cloning the resulting fragments into pGEM-T . The fragments were amplified using the following oligonucleotides (see Table S1 for sequences): 0.2 kb, ry-PvuI-F and ry-9350-R; 0.5 kb, ry-8981-F and ry-9532-R; and 1.0 kb, ry-8788-F and ry-9720-R.The 0.2-kb, 0.5-kb, and 1.0-kb donors were constructed by amplifying appropriately sized fragments from the 4.2-kb donor, using LA EcoRI and NdeI (removing the 5′-most 2.1 kb), blunting, and re-ligating the shortened vector.The 2.1-kb donor was constructed by digesting the 4.2-kb donor with AgeI and AflII, blunted with DNA polymerase I Klenow fragment , and re-ligated. This created a 347-bp deletion starting 269 bp downstream of the target site. The BX donor was constructed by digesting the 7.5-kb donor with XbaI and BspDI, then blunting and religating as above, creating a 1232-bp deletion beginning at the target site. The SA donor was constructed by digesting the 7.5-kb donor with AflII and SgrAI and blunting and religating as above, creating a 1307-bp deletion beginning 699 bp upstream and ending 608 bp downstream of the target site. The BP donor was constructed by digesting the 7.5-kb donor with BssHII and PmlI, then blunting and religating as above, creating a 1089-bp deletion ending 825 bp upstream from the target site. All donors were verified by sequencing at the University of Utah DNA sequencing Core Facility.Deletion donors were constructed using the 7.5-kb donor as a starting point. To construct the AA donor, the 7.5-kb donor plasmid was digested with Oligonucleotides were synthesized at the University of Utah DNA/Peptide Core Facility, except for those used for TaqMan analysis, which were made by Integrated DNA Technologies .in vitro from plasmids carrying the ryA and ryB ZFN coding sequences. The RNAs were mixed at 350 µg/ml each and injected into syncytial embryos by standard procedures. Each separate injection set involved approximately 200 embryos. Both plasmid and oligonucleotide donors were injected at a concentration of 500 µg/ml in the same solution as the ZFN RNAs. Some experiments with oligonucleotide donors were performed with TALENs directed to the ry target, rather than ZFNs . Experiments involving heat shock induction of ZFNs were carried out as previously described . If the deletion is present, the resulting fragment is 787 bp, while the corresponding wild-type fragment is 1130 bp. HR mutants were tested for the BX deletion by amplification with ry-PvuI-F and ry-10320-R. The wild-type fragment was 1396 bp; the deletion was 169 bp. To test for the BP deletion, a polymerase chain reaction (PCR) assay was performed with ry-506-7640-F and ry-PvuI-R in mutants that had already tested positive for HR. A wild-type fragment was 2230 bp; the mutant fragment was 1143 bp. SA deletions were identified by amplification with ry-NdeI-F and ry-genomic-R; the wild-type fragment was 2117 bp; the HR fragment was 810 bp.Mutations at the 1118w stock used for transformations of the heat shock-driven ZFNs. Five sites were chosen where a polymorphism changed a restriction site between the donor and the target chromosomes (see Table S1 for PCR primers). For each site, the region of the polymorphism was amplified and the resulting fragment digested with the appropriate restriction enzyme. The digested DNA was then visualized on an agarose gel and examined for either the donor or the target digestion pattern.Initial conversion tract experiments were performed in a stock that carried a third chromosome derived from the 169lig4 stock that lacked the BstNI polymorphism. An additional polymorphism was identified at position +3341 relative to the ZFN cut site between the donor and the ry locus in both the Canton-S and the 169lig4 stocks. In addition to restriction enzyme digestion, some loci were tested with alternative assays. The PvuII site at position −2812, the KpnI site at −460, the DpnII site at +391, and the site at +3341 were assayed using a customized TaqMan single-nucleotide polymorphism endpoint genotyping assay (Applied Biosystems) using an ABI 7900HT real-time detection instrument and analyzed with Applied Biosystems SDS version 2.3 software at the University of Utah Genomic Core. Primers for these assays were designed by the University of Utah Core facility. The HaeIII site at position +842 also introduces a 2-bp deletion in the target. PCR primers ry-842-FLP-F and ry-842-FLP-R (Table S1) were used to amplify fragments of 155 bp from the target and 157 bp from the donor. These were run on an Applied Biosystems 3130 × l capillary electrophoresis instrument  and analyzed with Applied Biosystems GeneMapper version 3.7 software. An additional polymorphism was identified between the donor and the 169 rylig4 locus at position +1985. This polymorphism was not present in the wild-type stock. The polymorphism introduces a 7-bp deletion in the target in comparison to the donor. The PCR primers ry-1985-FLP-F and ry-1985-FLP-R were used to amplify a 160-bp fragment from the target sequence and a 167-bp fragment from the donor. They were analyzed as described above.Some experiments with the 7.5-kb donor were performed in a Drosophila melanogaster. With one exception, ZFN mRNAs and donor DNAs were delivered by direct injection into embryos (All experiments were performed in rosy (ry) gene, and the corresponding ZFN pair was that described previously  covering most of the ry gene. New ry mutants among the progeny were identified by eye color. This procedure revealed only germline mutations and scored each parental ry allele individually. Thus, the proportion of offspring that carried new ry mutations in each experiment was equivalent to the frequency of mutation at the target. Many of the mutants were subjected to molecular analysis to distinguish HR products from those created by NHEJ. The designation % HR is the proportion of all mutants analyzed that scored as HR products.Injected flies were raised to adults and then crossed with partners carrying a deletion . We explored this limit in ZFN-induced HR in Drosophila by testing donors of various lengths incorporated into a circular plasmid with a backbone of approximately 3 kb . The homology in all cases, except that of the 4.2-kb donor, was approximately equally distributed on the two sides of the ZFN-induced break.Studies of homologous recombination in many organisms have shown that mechanisms depending on strand invasion require a minimum length of homology between donor and recipient. In eukaryotes, this lower limit is typically in the range of 200 to 400 bp  and is plig4 mutant, in the vicinity of 60% HR for homologies of 2.1 kb or longer. The % HR dropped to 24% for 1.0-kb and 0.5-kb donors and was undetectable with only 0.2 kb of homology.As shown in lig4 mutants than in the wild type . The latter represent only 3 of 68, 4 of 66, and 3 of 78 tracts in the three experiments and may be due to short-track mismatch correction in heteroduplexes formed during homologous recombination.Analysis of the conversion tracts individually showed that most were continuous, although a few showed interspersed donor and target markers .In the HS experiment, most conversion tracts were short, and the frequency of long tracts was quite low . In contFigure S3). These differences probably reflect the same phenomenon noted in the preceding paragraph, i.e., more of the tracts in the injection experiments showed complete conversion to donor sequence.One other feature differed between the heat shock and embryo injection data. In the injections, a larger proportion of the HR products showed two-sided conversion tracts, while from in the HS experiment, more were one-sided or simply involved the ZFN cut site  with the following characteristics: donor A consisted of 101 nt corresponding to the sense strand of the longer donors, carrying the substitution that replaces the zinc finger recognition sites with two in-frame stop codons and an XbaI site . In addition, the multiple mismatches between donors F and R and the target did not seem to affect their utility  large deletions in the donor DNA are readily incorporated at the target; and 4) single-stranded oligonucleotides serve as effective donors.In this study we examined in detail several parameters that affect donor utilization in ZFN-stimulated gene targeting experiments. Our principal findings were: 1) the minimum amount of homology necessary to support efficient gene replacement is between 200 and 500 bp with a circular donor DNA molecule; 2) conversion tracts are quite broad in events occurring in injected Drosophila. While the particular lessons derived are specific for this specific experimental system, the considerations are important for all nuclease-mediated targeting experiments. Because all events after target cleavage reflect activities available in cells for double-strand break repair, different systems will have different properties with respect to these parameters. In principle, gene targeting outcomes can be modified by manipulating relevant cellular activities, as we have done by using lig4 mutants to increase HR relative to NHEJ.Each of these conclusions is important for designing donor DNAs for future gene targeting applications in Drosophila, the frequency of NHEJ mutagenesis, the enhancement of HR in a lig4 background, and the utility of both plasmid and oligonucleotide donors reported here encourage the use of ZFNs for modifying essentially any genomic locus. The ryA:ryB ZFN pair is the most active of the ones we have tested, but we have achieved very good levels of targeted mutagenesis at other loci by using designed ZFNs . In the embryo injection system, homology lengths greater than 1 kb provide additional efficiency, particularly in a lig4 mutant background.The minimum length of matched sequence required for efficient homologous recombination has sometimes been called minimum effective processing segment (MEPS) . It is uAs has been demonstrated in mammalian cells   carried a deletion of 23 bp and an insertion of 12 bp relative to the target sequence.Consistent with our analysis of conversion tracts based on very localized polymorphisms, we also found that deletions of hundreds of base pairs were readily converted from donors into the With the oligonucleotide donors, the efficiency of HR was somewhat lower for the version carrying the deletion/insertion opposite the cut site than for the one with a single base pair deleted . This obry expression , expression of the inserted gene was apparently suppressed due to natural regulation of


Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector. Poxviridae are variola virus, the causative agent of smallpox, and vaccinia viruses (VACV), the smallpox vaccine strain. Smallpox was declared eradicated in 1979 by the World Health Organization (WHO) following a long, global vaccination campaign Poxviruses are cytoplasm-replicating large double-stranded DNA viruses. Two of the best-known members of the family of http://www.virology.ca). Several reports have illustrated the presence of genomic diversity in these earlier generations of smallpox vaccines. Qin et al., sequenced 11 clones isolated from Dryvax and revealed the presence of genetic diversity, with deletions in the right-hand inverted terminal repeat (ITR) Currently, there are 30 VACV genomes stored in the Viral Bioinformatics Resource Center (www.IAVI.org). In this study, we report the results from sequencing and virulence characterization of TT clones derived from TT (752-1).The original TT strain was previously reported to have been isolated from skin lesions of a Chinese individual with smallpox in 1926 and had an attenuation history of passages in monkey, rabbit, bovine skin and rabbit testis 2 atmosphere. TT clones were isolated from TT (752-1) by plaque-purifications at terminal dilutions. Seven viral plaques were isolated and amplified by sequential four rounds of passages to produce enough material for sequencing TT (752-1) was provided by the National Vaccine and Serum Institute, Beijing. NYCBH strain was provided by the AIDS Research and Reference Reagent Program  of the U.S. National Institutes of Health. Both viruses were grown in CEF prepared from 9 to 11 day-old embryonic chicken eggs and cultured in Eagle’s medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% antibiotic at 37°C in a 5% COThe genomes of TT clones were sequenced by Illumina Hiseq2000 Sequencer  using shotgun sequencing of 500 bp paired-end sequencing library. Contigs were assembled and scaffolds were constructed using raw data with SOAP denovo and SOAP aligner (Beijing Genomics Institute) . In cases where an ORF contained more than one start codon (ATG) near the 5′ terminal, BLAST was conducted to compare the orthologous sequences in other VACVs. The most frequently used ATG was selected as the tentative ORF in TT.Genome Annotation Transfer Utility (GATU) http://athena.bioc.uvic.ca/virology-ca-tools/base-by-base/) http://www.atgc-montpellier.fr/phyml/). The phylogenetic tree was visualized using Figtree (http://tree.bio.ed.ac.uk/software/figtree/). MEGA 5 BioEdit was used to produce alignments of multiple genomes by Clustal W method To analyze the genomic sequence orthology among TT clones, a consensus sequence was first derived. TT11 had the highest similarity to the consensus sequence and was therefore chosen as the reference. ORFs of TT11 were compared with orthologous ORFs of four other TT clones and other VACV genomes to calculate the full-length nucleotide sequence similarity using GATU and DNAMan .The full genomic sequences of five TT clones were deposited in GenBank with accession numbers from JX489135 to JX489139. The OPVs used in this report and their GenBank accession numbers are listed in 2 plaque forming units (PFU) in 30 µl PBS were inoculated intracranially to groups of five 3-week old female BALB/c mice anesthetized with 1.25% 2,2,2-tribromoethyl alcohol. Body weights and mortality were recorded daily during the 12-day observation period.TT viruses and the NYCBH strain were serially diluted in phosphate buffered saline (PBS) and then 3 x 106 to 102 PFU/ml in PBS. For each viral dilution, two rabbits were tested. Each rabbit received two dorsal skin intradermal inoculations with 0.1 ml viral preparation per injection site. At day 4 post-infection (p.i.), the diameters of lesions were measured and the incidence of putrescence was recorded.TT clones and the NYCBH strain were serially diluted from 10All experiments were conducted in accordance with the guidelines of the Laboratory Animal Center of Chinese Center for Disease Control and Prevention and NCAIDS. All procedures involving animal use and care were approved by the Institutional Committee on Laboratory Animals of NCAIDS.Seven randomly selected TT clones were isolated by plaque picking at the terminal dilution on CEF cells and amplified. The sizes of plaques varied after initial runs of plaque picking of each clone. No obvious difference was observed between the plaque sizes of the clones following passage and those of the original vaccine pool, although the average plaque sizes of TT9 and TT11 were significantly smaller (p<0.05) than those of TT7 and TT8 .All seven viral genomic DNAs were sequenced and their contigs were assembled. All the assemblies were aligned with Cop and ListerV107 to accurately identify any gaps, particularly in highly repeated elements such as the ITRs. Five TT clones (TT8 to TT12) were successfully assembled and generated full genomic sequences of 189,366 to 191,144 bp. Two clones, TT6 and TT7 had small gaps in the two ITRs and thus did not yield full genomic sequences.We examined the phylogenetic relationship of various OPVs by maximum-likelihood method using alignments of the conserved 160.4 kb central region  . These sNotably, TT clones clustered with a bootstrap value of 988 and shared a common ancestry with the TT genomic strain (AF095689.1) published in 1998 and another TT (752-1) derived TT-TW sequenced in 2010. Among the other VACVs, Cop was most similar to the TT cluster and HSPV-MNR76, NYCBH/Dryvax-derived DPP clones, Acam2000 and Duke exhibited least similarity. Among the analyzed OPV sequences, ectromelia virus strain Moscow (ECTV-Mos) was most distant from the TT cluster.All the Dryvax derived strains clustered as one group, including the Dryvax clones (DPPs), Acam2000 that were independently isolated from another stock of the same vaccine, and Duke that isolated from a patient experiencing a Dryvax vaccine-associated complication. The TT clones exhibited higher sequence diversity than the Dryvax clones. The average value of pairwise distances between TT clones was 0.00401 (σ = 0.00026) and the average value between Dryvax clones was 0.00294 (σ  = 0.00017).ORFs longer than 50 aa were initially annotated as genes and those without comparable orthologs in other OPVs were removed from annotation. Small ORFs that embedded in larger ORFs in the genomes of other poxviruses and those found to be truncated or split into two or more portions were removed from the ORF list and reclassified as “fragments”. Six such fragments were identified: TT_005.1, TT_028.1, TT_191.1, TT_210.1, TT_252.1, and TT_265.1. The genomic organizations of TT clones were identical to other sequenced VACVs. There were 255 unique ORFs in the genome and nine duplicated ORFs (TT_001 to TT_009 and TT_261 to TT_269) in ITRs. There are 273 ORFs found in each of the TT clones , includiThe orthology between TT clones was further assessed through the distribution of polymorphic sites. We first generated a consensus sequence using the most frequent nucleotide from each position. The similarities of each TT clone varied from 97.36% to 98.30% and TT11 had the highest similarity with the consensus . The ORFSeveral genes had polymorphic lengths because of the presence of substitution mutations. As shown in VACV contains more than 200 genes, some of which have been previously reported to be associated with viral virulence and immunomodulation. We selected seven virulence reaction-associated genes from TT clones and Acam2000 for amino acid sequence comparison . Acam200Six immunomodulation-associated genes were also selected for closer examination . TT_210 2 PFU viral preparations or PBS and observed daily for weight change and mortality. In the control group (injected with PBS), all mice gained weight over the 12-day observation period. Mice that received NYCBH showed severe signs of illness, weight loss , TT clones, and NYCBH. Groups of three-week-old BALB/c mice were intracranially inoculated with 3 x 10ght loss , and decght loss . By day We examined the virulence of five TT clones in the same manner. The patterns of weight loss  and mort2 to 105 PFU, into rabbit dorsal skin. Viral virulence was quantified by the diameter of skin lesion on day 4 p.i. As shown in 5 PFU. Among TT strains, the parental strain yielded the largest lesions at the dilution of 105 PFU (4.6 mm) followed by TT8 (4.2 mm), TT9 (3.4 mm), TT10 (3.4 mm), TT12 (1.2 mm), and TT11 (1.0 mm). NYCBH yielded 100% putrescence at all dilutions. TT clones yield 100% putrescence at the highest 105 PFU inoculation, much less putrescence at the 104 PFU inoculation, and no putrescence at the 103 PFU inoculation.We further characterized the skin virulence of the parental and cloned strains by inoculating four viral preparations, 10Similar to most vaccinia viruses used in the last century, TT has a vague origin and passage history. The earliest record shows its ancestral strain was isolated from skin lesions of a Chinese individual with smallpox in 1926 The genome structure of TT is the same as other vaccinia viruses. The genome length of TT clones varied from 189,366 in TT8 to 191,144 in TT11 with 273 identifiable ORFs. Insertions, deletions, and point mutations were present throughout the genome resulting in protein alterations. ORFs located in the middle of the genome were more conserved than those located at the two ITRs. We also examined biological activities of TT strains in mice using intracranial injections. TT (752-1) and its progeny clones behaved different in the induction of weight loss and mortality. These biological variations among TT clones may not be due to the replication abilities since they were similar in our study (data not show). The severity of the weight loss and mortality was less than that produced by the NYCBH strain. Our finding differs from Marennikova et al. who reported that TT displayed higher pathogenicity than NYCBH in mice Our present genomic and phenotypic characterization of TT strains yields valuable information for continuing TT-based research in China or elsewhere to produce safer and more effective smallpox vaccines. It is also useful for the current vaccine research using TT-based vectors for HIV and other pathogenic viruses.Figure S1Plaque properties of TT (752-1) and seven TT clones. Each clone of the TT (752-1) was plaque-purified and ∼50 PFU of each clone was plated on a monolayer of CEF cells. The infected cells were cultured for 3 days and then stained with crystal violet and imaged. The plaques were visualized using ImageJ and their sizes were determined.(TIF)Click here for additional data file.Table S1Identities and GenBank accession numbers of orthopoxviruses cited in this work.(DOC)Click here for additional data file.Table S2Complete complement of genes and gene fragments encoded by TT clones. a: The orthologs in Cop and WR were used as the reference. When no ortholog was present in these two reference strains the gene with the highest similarity in OPVs was used. b: orthologs of "X_ORF_X" in Cop were annotated in TT clones. These genes were predicted not to express. c: TT_005.1, TT_028.1, TT_191.1, TT_210.1, TT_252.1, and TT_265.1 were identified as fragments.(DOC)Click here for additional data file.Table S3Full length similarities among nucleotide sequences of orthologs in TT clones and four reference strains to TT11. TT11 was used as the reference clone for comparison with TT8, TT9, TT10, TT12, and four other VACVs .(DOC)Click here for additional data file.


ET scale for ionic liquids neglect this mutual influence, which, however, seems to be in fact necessary to get a proper description of ionic liquid properties. It is shown how strong such parameters vary when the influence of the counter ion is taken into account. Furthermore, acceptor and donor numbers for ionic liquids are presented.Solute properties are known to be strongly influenced by solvent molecules due to solvation. This is due to mutual interaction as both the properties of the solute and of the solvent strongly depend on each other. The present paper is based on the idea that ionic liquids are cations solvated by anions and anions solvated by cations. To show this (in this system strongly pronounced) interaction the long time established donor–acceptor concept for solvents and ions in solution by Viktor Gutmann is extended to ionic liquids. A number of solvent parameters, such as the Kamlet–Abboud–Taft and the Dimroth–Reichardt  ET polarity scales. AN and DN were determined by spectroscopic methods (UV/Vis) using the solvatochromic indicators Fe(phen)2(CN)2 for AN and Cu(acac)(tmen)+ for DN. SolvatochromismThe selection of an appropriate solvent or solvent mixture to perform chemical reactions or to separate mixtures of reactants and products is crucial in many chemical processes.r as correlation factor:The determination of AN and DN for solvents is carried out straightforward by dissolving the appropriate indicator and measuring the shift of the absorption maximum in the respective solvent to a reference solvent .The determination of donor and acceptor numbers for ions in non-aqueous solvents is more complex. The ability of anions (cations) to act as an electron pair donor (an electron pair acceptor) influences the donor and acceptor properties of a solvent. The interaction of the cations with the iron indicator depends on both the acceptor and donor properties of the solvent. If the solvent is a stronger Lewis acid than the cation, the solvent interacts solely with the indicator, or the solvent is a Lewis base strong enough to solvate the cations making an interaction between indicator and cation impossible. In these two cases no significant change in the absorption maximum is found as the influence of the dissolved cation is negligible (solvent dependent part). An increase of Lewis acidity and therefore an increase of acceptor properties of the cation leads to a shift of the absorption maximum to lower energies (cation dependent part) . At the intersection point of the solvent dependent part with the cation dependent part, both the acceptor properties of the solvent and those of the cation are equal and the acceptor number thus obtained is defined as the apparent acceptor number of the cation dissolved in that particular solvent :A multiple regression analysis of the data yields for The interaction of the anions with the copper indicator also depends on both the acceptor and donor properties of the solvent. If the donor strength of the solvent is higher than that of the anion the solvent coordinates preferably with the copper complex, however, if the acceptor strength of the solvent is too high it will compete with the indicator for the coordination of the anion and no change in the absorption maximum is found (solvent dependent part). If the donor strength of the anion exceeds the donor strength of the solvent a shift of the absorption maximum can be observed (ion dependent part). For strong acceptor solvents such as water and methanol even strong donating anions cannot coordinate to the copper complex as they are preferentially solvated by such solvents . At the intersection point of the solvent dependent part with the anion dependent part, both the donor properties of the solvent and those of the anion are equal and the donor number thus obtained is defined as the apparent donor number of the anion dissolved in that particular solvent :A multiple regression analysis of the data yields for The same concept for ions in non-aqueous solutions shall be applied for the determination of donor and acceptor numbers of ionic liquids. As stated above cations and anions influence the donor and acceptor properties of a solvent thus shifting the absorption maximum of a solvatochromic dye. In the case of ionic liquids the assumption is made that cations are solvated by anions and anions solvated by cations. The anions therefore are acting as solvent for the cations and the cations as solvent for the anions. Therefore there is again a mutual influence of the ions on their donating and accepting ability. A system of equations can be established in order to reflect the ionic influence:a, b and A, B), donor and acceptor numbers for neat ionic liquids can be obtained. Using again dichloroethane and nitromethane as reference solvents the DN and AN are on the same scale as Gutmann’s DN and AN for solvents.By solving the system of Equations (7) and (8), using the previously determined constants  based ionic liquids. Figure H value of the reaction of SbCl5 with a solvent molecule L to the adduct SbCl5⋅L in high dilution of DCE. Here DCE has been defined as reference solvent with DN=0 . For commonly used solvents this reaction is exothermic so that ΔH turns out to be positive and usual values are in the range between 0 (DN) and 100. AN on the contrary is defined as the NMR chemical shift of the 31P nucleus in Et3PO in the respective solvent (undiluted). To obtain comparable values with the DN scale this NMR shifts (measured in ppm) have been renormalized to become 100 for the (DN-important) probe SbCl5. Negative AN (DN) just means, that the ionic liquid in question is a weaker acceptor (donor) than nitromethane (dichloroethane). For example, in acetone (AN=12.5) Cl− is as strong donor (DN=38.5); upon changing to a solvent with a higher AN the donicity of Cl− shrinks (DN in methanol is only 22.6). In [C4C1im][Cl] the solvation shell consists of C4C1im+ cations which are accepting most of the electrons from Cl− and the cation is therefore a very weak acceptor and the Cl− is a weaker donor than in acetone or methanol. [BF4]− exhibits in acetone a donor strength of 8.33 and in methanol −7.56; in C4C1im+ the DN is −4.18, having approximately the same donicity as [CF3SO3]− in water (DN=4.0).16Correlation between DN and NMR shifts: It has been shown that the Kamlet–Abboud–Taft solvent parameter β  ability) is linearly correlated to the H-2 hydrogen NMR chemical shift 4C1im+ based ionic liquids.4C1im+ is fixed. Any alteration on the electron density is therefore dependent on the nature of the anion.4C1im+ based ionic liquids where a solvatochromic approach is not feasible :k1=8.4667, d1=−73.7101, k2=−4.1043 and d2=−9.7339. Table 4C1im+ ionic liquids determined by solvatochromic indicators. In the series of chloroaluminate anions it can be seen, that an ionic liquid with a low mole fraction of added AlCl3 exhibits strong donating abilities, whereas the donor properties are reduced as the mole fraction rises .As there is no additional NMR sensitive probe molecule in the ionic liquid (the shift of a NMR probe such as triethylphosphine oxide should be dependent on donor and acceptor properties as do the solvatochromic indicators) the donor properties of the anion can be screened solely by the shift of H-2, that is, the H-2 proton acts as “built-in” indicator.Cation variation: Figure 1,1,1,4+, S2,2,24+, C4py+, C1C4pyrr+, C2C1im+, C4C1im+, C6C1im+ and C8C1im+) give a steeper slope than cations with a high steric demand . Although choline is not sterically demanding it does not correlate with the other less sterically demanding cations. The reason for this could be the functional group OH which gives an additional possibility of interaction  with a given anion. Table Side chain length variation: The interpretation of the influence of the side chain length is not straightforward. Considering a virtual, from anions unaffected Cnmim cation, a linear decrease of AN would be expected due to the increasing inductive effect of a longer side chain. From the available data the results show local minima and maxima for DN and AN, respectively, in dependence of the chain length. The DN and AN values are still linearly correlated but not in the order as it would be expected from the increasing inductive effect  of the probe triethylphosphine oxide is dependent on the solvent used. Using conventional solvents the donor property of a solvent is negligible, but not in ionic liquids, were the solvation shell of the probe molecule coordinated to the cation (Lewis acid) consists of anions (Lewis base). Therefore all previously reported acceptor numbers are too high since the lowering effect of the anion is not considered. To visualize the importance of mutual interactions to acid–base considerations, one might compare AN and DN (representing Lewis acidities and basicities) with “simple” Brønsted relations. Taking H3BO3 for example, which is a very weak acid an aqueous solutions  becomes strongly deprotonated in liquid ammonia and is acting in reactions accordingly in such media. On the other hand, strong bases will lose their basicity in liquid ammonia. The situation obviously becomes reversed using acidic media such as water-free acetic acid as solvents.Acceptor numbers determined from Kamlet–Abboud–Taft parameters,α and β, but also a function on the anion (cation). Increasing the basicity, for example, from [BF4]− to [Cl]− the accepting ability is reduced. Increasing the acidity, for example, from + to [chol]+ the donating ability of [NTf2]−is reduced.These results indicate that ionic liquid cations and anions cannot be discussed separately; the mutual influence of the counter ion has to be considered; this means that the acceptor (donor) properties are not only a function of the cation (anion), as it is usually suggested for the Kamlet–Abboud–Taft parameters 4C1im+ based ionic liquids are usually compared to lower alcohols.Frequently the polarity of ionic liquids is described to behave like conventional solvents2T  and the Institute of Applied Synthetic Chemistry  or were prepared according to literature protocol.4] or [Fe(phen)2(CN)2]) dissolved in dichloroethane (DCE) is added to 400 μL ionic liquid dissolved in DCE. This ensures a homogeneous distribution of the indicators. The solvent is then removed at reduced pressure (approx. 10−3 mbar). The so prepared samples are transferred into a cuvette  and are measured against the pure, dried ionic liquid. UV-Vis spectra were obtained on a Perkin–Elmer Lambda 900  or a Carl Zeiss MCS 400  spectrophotometer in transmission mode at room temperature. The spectral range was 300 nm to 1000 nm.The ionic liquids used in the study were purchased from Sigma–Aldrich  and TCI  or were a donation from AC


In vivo, G-CSF impaired apoptosis induced by oxidized low density lipoprotein (OX-LDL) but it had little effect on cultured endothelial cells (ECs) with vehicle treatment. Further research revealed that G-CSF promoted the upregulation of endothelin-1 (ET-1) and the downregulation of endothelial nitric oxide synthase (eNOS) of thoracic aortae induced by a high-fat diet. In vitro, the effects of G-CSF on expression of ET-1 and eNOS in cultured ECs were consistent with those in vivo. Our results suggested that G-CSF exacerbates lipid abnormity and endothelium damage in hyperlipidemia rabbits, thereby resulting in the deterioration of atherosclerosis and that the ET-1/eNOS system may regulate the progression.Granulocyte-colony stimulating factor (G-CSF) has been reported to improve the function of infarcted heart, but its effects on atherosclerosis are unclear. Here we examined the effects and the potential mechanisms in the high-fat diet rabbit model. Six-month-old male New Zealand white rabbits, fed a high-cholesterol diet or a normal diet for 10 weeks, were treated with vehicle or G-CSF. G-CSF increased lesion area in the thoracic aorta and the plasma levels of total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) at the early phase in the high-fat diet group. High-fat diet-induced arterial endothelium damage and apoptosis were greatly aggravated by G-CSF treatment.  Granulocyte-colony stimulating factor (G-CSF) has been shown to repair the infarcted heart not only by inducing mobilization of bone marrow stem cells  but alsoIn this study, to elucidate whether the effects of G-CSF on the development of atherosclerosis are different between subjects with or without hyperlipidemia, we used rabbit models, fed a normal or high-fat diet and examined the atherosclerosis in the thoracic aortae of these animals with or without G-CSF treatment.G-CSF has been reported to improve the function of infarcted heart, but its effects on atherosclerosis are unclear. Here, our results show that G-CSF promotes atherosclerosis in rabbits with a high-fat diet. The inner surfaces of the aortae of the normal diet group with or without G-CSF treatment were smooth and flat, and showed no positive Sudan III staining . In the et al. recently reported that G-CSF had no adverse effects on atherosclerotic lesions in high cholesterol-fed miniature swine [However, G-CSF has been observed to prevent the progression of atherosclerosis in MI and balloon injury rabbits in the study of Hasegawa H . The prore swine , the difre swine .Hyperlipidemia is well recognized as an important risk factor for atherosclerosis , and hypIn the present study, G-CSF did not affect the plasma lipids in the normal diet group, but it increased plasma TC and LDL-C in the high-fat diet groups at the early phase although it had little effect on the peak concentration of plasma lipid. Haghighat reported that G-CSF did not affect serum lipids in apolipoprotein e-deficient mice . HoweverVascular endothelial cell (VEC) injury represents a major initiating step in the process of atherosclerosis. Normal endothelium is antithrombogenic, and if injured, it promotes platelet aggregation and coagulation. Endothelial damage can cause vasospasm, intimal hyperplasia, and arteriosclerotic acceleration . In our in vitro and in vivo which may result in a worsening effect on atherosclerosis.In order to examine whether G-CSF exerts a synergestic effect on apoptosis in endothelial cells, we next performed TUNEL analysis on thoracic aorta tissue. In the lesion area of the thoracic aorta, ECs were seriously damaged, so we analyzed the data in the non-lesion area. The results revealed that G-CSF significantly increased TUNEL-positive ECs in the non-plaque area of the thoracic aorta in high-fat diet rabbits, but it did not induce apoptosis of ECs in normal diet rabbits . We nextin vivo. As an index for the impaired endothelial function, ET-1 exacerbates endothelial dysfunction to some extent [A previous study reported that ET-1 exacerbated endothelial dysfunction  and thate extent . Endothee extent . So G-CSThus, our data revealed that G-CSF deteriorated atherosclerosis by promoting lipid abnormity at the early phase and endothelial cell dysfunction by the ET-1/eNOS system in high-fat diet rabbits. In most clinical trials, five to six total doses of G-CSF were given over a one-week period. G-CSF may be a risk factor for exacerbating atherosclerosis especially in patients with hyperlipidemia. The doses of G-CSF used in the present study were similar to those used in humans. However, the dosing schedule was more frequent in that the animals received a total of 15 doses over a three-week period. Significant effects of G-CSF on the atherosclerotic lesion area were seen only in animals that also received a high-fat diet. It suggests that the increased course of G-CSF treatment may produce a deleterious effect on atherosclerosis especially in patients with the hyperlipidemia state. Our limitation is that we neither compared the effect of G-CSF on atherosclerosis at different frequencies nor explored other potential mechanisms. Randomized clinical trials to evaluate the feasibility and safety of G-CSF on coronary artery diseases induced by hyperlipidemia are warranted.n = 6); normal diet plus G-CSF group (n = 8); high-fat diet plus vehicle group (n = 8) and high-fat diet plus G-CSF group (n = 8). The normal diet groups were fed normal rabbit chow, whereas the high-fat diet rabbits were fed a diet containing 1% cholesterol and 5% lard for 10 weeks. In G-CSF-treated groups, recombinant human G-CSF  was injected subcutaneously for the first 3 weeks . The same volume of saline was injected subcutaneously as control. All protocols were approved by the Institutional Animal Care and Use Committee of Fudan University, and in compliance with “Guidelines for the Care and Use of Laboratory Animals” published by the National Academy Press [The 6-month-old male New Zealand white rabbits  were randomized into four groups: normal diet plus vehicle group , triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) and high cholesterol density lipoprotein-cholesterol (HDL-C) and the values of circulating white blood cells (WBC) were measured by the standard method in the Department of Laboratories in Zhongshan Hospital, Fudan University.The animals were sacrificed at the end of study and the thoracic aorta was excised for morphological analysis as follows: (1) Sudan III staining: the thoracic aortae cut to less than 2 cm were fixed by normal 10% neutral formalin and stained by Sudan III. The degree of atherosclerosis was evaluated as the percent of lesion area of the total surface area of the intima (the surface area of lesions/the surface area of the whole intima) with the Leica Qwin V3 analysis system. (2) Paraffin-embedded thoracic aorta tissues were subjected to hematoxylin-eosin (HE) staining by the standard procedure. (3) For scanning electron microscopy examination, thoracic aorta tissues were fixed in 2.5% glutaraldehyde, post-fixed for 1 h in 1% osmium tetroxide, rinsed with distilled water, dehydrated and then observed under a HITACHIS-520 scanning electron microscope. (4) Transmission electron microscopy analysis: thoracic aorta sections were fixed in 4% paraformaldehyde, post-fixed in 1% osmium tetroxide solution, dehydrated and embedded in epoxy resin. Semi-thin (1 μm) serial sections were prepared and ultra-thin sections (50 to 70 nm) were mounted on Formvar-coated  75 mesh copper grids and counterstained with uranyl acetate and lead citrate before analysis under a JOEL-1200 transmission electron microscope.5/well. These cells were pretreated with G-CSF (50 ng/mL) or vehicle for 30 min followed by stimulation with or without oxidized low density lipoprotein  for 24 h. Finally, these cells were collected for further TUNEL analysis and real-time RT-PCR analysis.Aortic endothelial cells were isolated from the thoracic aortae of rabbits by a previously described method . The endin situ cell death detection kit. Merck Inc., CA, USA). The cells, grown on glass coverslips, were incubated with 50 μL TUNEL reaction mixture containing TdT for 60 min at 37 °C. DAB substrate solution was then added, and the mixture was incubated for 10 min at room temperature. The apoptotic cells were expressed as a percentage of TUNEL positive cells of all the cells obtained from the slides.TUNEL labeling was performed according to the manufacturer’s protocol . GAPDH was used as an internal control to normalize for RNA amounts. The primers used were as follows: GAPDH, upstream primer, 5′-CCACTTTGTGAAGCTCATTTCCT-3′, downstream primer, 5′-TCGTCCTCCTCTGGTGCTCT-3′; ET-1, upstream primer, 5′-CTCTCTGCTGTTGGTGGCTTT-3′, downstream primer, 5′-TGGGTTTCCGCTCCTGT-3′; eNOS, upstream primer, 5′-AGGCCTCCTGTGAGACTTTC-3′, downstream primer, 5′-AAGGAGTCGAGGACTGGATG-3′.p-values < 0.05 were considered statistically significant. Statistical analyses were performed by the SPSS 13.0 software package .Data are expressed as mean ± SEM. The comparisons among groups were tested by one-way ANOVA. Our results suggest that G-CSF and hyperlipidemia exert synergistic effects on lipid abnormity and endothelial cell dysfunction, which resulted in the deterioration of atherosclerosis in high-fat diet rabbits. The ET-1/eNOS system may regulate the progression. Therefore G-CSF may be a risk factor for exacerbating atherosclerosis especially in hyperlipidemic patients.


Common uses of the granulocyte-colony stimulating factors in the clinical practice raise the concern about side effects of these agents. We presented a case report about an acute myocardial infarction with non-ST segment elevation during filgrastim administration. A 73-year-old man had squamous cell carcinoma of larynx with lung metastasis treated with the chemotherapy. Second day after the filgrastim, patient had a chest discomfort. An ECG was performed and showed an ST segment depression and negative T waves on inferior derivations. A coronary angiography had showed a critical lesion in right coronary arteria. This is the first study thats revealed that G-CSF can cause acute myocardial infarction in cancer patients without history of cardiac disease. Patients with chest discomfort and pain who are on treatment with G-CSF or GM-CSF must alert the physicians for acute coronary events. Granulocyte-colony stimulating factors (G-CSF) are commonly used in patients with chemotherapy-induced neutropenia. Recently G-CSF has been used in clinical trials to research neovascularization and/or to reduce the damaged size of infarct. Common uses of the granulocyte-colony stimulating factors in the clinical practice raise the concern about side effects of these agents. Studies showed that nearly 5% of patients undergoing peripheral blood stem cell mobilization with G-CSF developed venous thromboembolic events (VTEs) . In addiA 73-year-old man had squamous cell carcinoma of larynx with lung metastasis treated with the chemotherapy including the docetaxel, cisplatin, and fluorouracil regimen. The patient was admitted to the hospital for pneumonia after the 3rd course of chemotherapy. Piperacillin tazobactam of 4∗2.25 gr per day was given. On the 4th day of the treatment of antibiotic, patients become neutropenic without fever, and then filgrastim 5 mcg/kg/day was administered. Patient had a chest discomfort on the second day of filgrastim administration. An ECG was performed, and ST segment depression with negative T waves on inferior derivations of the ECG had been determined . PatientThere have been many case reports about the thrombotic events in cancer patients receiving chemotherapy. Most of the case reports were associated with granulocyte-macrophage colony stimulating factors (GM-CSF), but there are no clear data in the literature about G-CSF. We presented a myocardial infarction case during the G-CSF treatment in a patient without history of coronary heart disease. G-CSF and GM-CSF have cardiovascular adverse events. Tolcher et al. had reported 2 cases about the iliac arterial thrombosis related to the GM-CSF, and Waldecker-Herrmann et al. also reported a catheter-related thrombosis of the internal jugular and subclavian vein during GM-CSF , 4. EckmThis is the first case report that revealed that G-CSF can cause acute myocardial infarction in cancer patients without a history of cardiac disease. In cancer patients, physicians must alert for acute coronary events when a patient has chest discomfort or chest pain especially during colony stimulating agent treatment.


The pre-clinical diagnostics is essential for management of Parkinson’s disease (PD). Although PD has been studied intensively in the last decades, the pre-clinical indicators of that motor disorder have yet to be established. Several approaches were proposed but the definitive method is still lacking. Here we report on the non-linear characteristics of surface electromyogram (sEMG) and tremor acceleration as a possible diagnostic tool, and, in prospective, as a predictor for PD. Following this approach we calculated such non-linear parameters of sEMG and accelerometer signal as correlation dimension, entropy, and determinism. We found that the non-linear parameters allowed discriminating some 85% of healthy controls from PD patients. Thus, this approach offers considerable potential for developing sEMG-based method for pre-clinical diagnostics of PD. However, non-linear parameters proved to be more reliable for the shaking form of PD, while diagnostics of the rigid form of PD using EMG remains an open question. Parkinson’s disease (PD) is a progressive disorder which affects motor, higher mental and autonomic functions of the human organism. PD is the second most common, after Alzheimer’s disease, neurodegenerative disease. The incidence of PD in developed countries is estimated at about 1% in people older than 60 years and 4% in people over 80 years  and non-motor symptoms . These motor and non-motor symptoms may decrease person’s ability to work and thus may imply early retirement, restrictions on profession choice and, hence, the salary . Cost buParkinson’s disease diagnosis is highly uncertain in the early stages and only 70% of patients are correctly diagnosed with PD . PD is cPD is clinically not uniform and is presented by at least three clinical forms – tremulous-dominant, or shaking form, akinetic-rigid form and mixed form. The incidence of tremor-dominant and mixed (tremor plus akinesia/rigidity) type of PD is as much as 75% .Parkinson’s disease may provide insight into such phenomena of the motor system as muscle tone, posture, gate and tremor, and yet enigmatic “motor commands,” and “motor programs.” For example, a characteristic spine bent “posture of beggar” in PD patients is analogous to the “tired ape” stance of astronauts/cosmonauts first described by Edwin Aldrin during Lunar mission  or to thSubstantia nigra. When approximately 60% of these are lost, PD becomes clinically recognizable. This phase is believed to start long before emergence of clinical PD and then slowly progresses usually over 4.5 years , ultrasound, and genetic identification. The other approach would be to detect PD patients at the very initial phase of the disease when only few non-motor or “soft” motor symptoms are detectable.Parkinson’s disease is characterized by a lengthy prodromal, which is known as either “pre-clinical” or “pre-diagnostic” phase , 12. The.5 years , 14. Bes.5 years . As such.5 years . Approxi.5 years . Therefo.5 years , 18. Thu.5 years  suggest Thus, pre-clinical detection of PD seems to be an important goal because even subtle motor or non-motor pre-clinical abnormalities may serve as “predictors” for further PD. Such predictive study would help identifying the subjects at-risk of future PD, to start earlier anti-PD treatment, and to develop effective neuroprotective treatment strategies . Here, wPatients with PD have several symptoms other than motor ones (the non-motor symptoms). Few of them have been proposed for early detection of PD. Among them are: (1) olfactory disorder; (2) sleep disorder; (3) autonomic features (heart rate variability and constipation); (4) color vision disorders; and (5) so-called “soft” signs of PD such as reaction time slowing, depression, mid-life obesity, and non-specific pain in joints . These dOlfaction is impaired first in PD due to affection of the olfactory bulb, thus forming stage I of PD . Smell dAutonomic symptoms, such as constipation and heart rate variability, and affective symptoms  are also candidates for early stage PD diagnostics , 19, 25.Basal ganglia seen on MRI is often associated with neurodegenerative diseases other than PD  , positron emission tomography (PET), fMRI and transcranial sonography are widely used to assess dopaminergic function in PD patients, their relatives, and healthy controls . Howeveratrophy) . Also, matrophy) .In a whole, definitive evidence for a PD-sensitive diagnostic tool is lacking. Combination of the above mentioned methods would probably be the best current solution for pre-clinical diagnosis of PD. The decisive diagnosis of PD is still post-mortem. All in all, current methods help to diagnose only 70–80% of the PD cases , 31, whiElectromyography (EMG) helps investigating the central nervous system (CNS) because it reflects the activity of the spinal motoneurons due to motor units (MUs). We seek to provide a readable method to diagnose PD based on EMG. In ideal, such method would also be helpful to detect PD either at early stage or even pre-clinically. Several studies have reported that MUs in PD patients discharge with alternating shorter and longer interspike intervals (doublets or triplets). This pattern of activity is strikingly different from stationary activity of MUs under normal muscle tone –34. NoneThere has been an attempt to combine EMG with thermal interventions as a provoking factor for PD symptoms. Cold was reported to intensify tremor in PD patients, especially with tremor-dominated form, while heat reportedly attenuated muscle rigidity . NonetheIn contrast to needle EMG, surface EMG (sEMG) is non-invasive (less discomfort and risk of infection), more stable in respect with electrode position (more repeatable), and cost-efficient. sEMG has been extensively used to examine motor function and movement disorders in humans and it is believed to provide relevant information on neuromuscular strategies . SpectraThe morphology-based analysis has shown promising results in discriminating PD and healthy controls. The method is based on the histogram and crossing rate analysis of sEMG signals . sEMG kuPrevious works have reported that sEMG waveform can better be modeled as an output of a non-linear dynamic system, rather than a stochastic output of a white-noise driven linear system . Non-linsEMG in PD patients is known to be rich in regular clusters (grouping) at the characteristic tremor frequencies (4–6 Hz) due to increased synchronization of MU . Also, dn = 30) and two healthy control groups of different age – young (n = 20) and old (n = 20). sEMG was recorded bilaterally in the upright stance from biceps brachii muscles under elbow flexion. The loading conditions were 0, 1, and 2 kg respectively. Complexity and regularity of SEMG was analyzed by various methods of non-linear time series analysis, including sample entropy (SampEn), correlation dimension (CD), percent of determinism (DET%), and recurrence rate (REC%) based on RQA. The amplitude of sEMG was defined as the root mean square (RMS) value and median frequency (MDF) was also determined for analysis.This subsection deals with our previous study , in whicThe major finding of our studies was that non-linear parameters of sEMG signal in the PD group significantly differed from the ones in the healthy control groups . In partDecreased CD of sEMG in the PD group may indicate increased self-similarity of the myoelectrical signal over time  and hencR = 0.47–0.71) (R = 0.54–0.66)  . It coulIn another previous study by our group , we measAcceleration signal demonstrated a very much the same correlation with loads, UPDRS, and motor symptoms as the sEMG signal . HoweverR = 0.47–0.52) and finger tapping (R = 0.32–0.46)  . Thus, a32–0.46) . Figure –0.46 . TNo major difference in sEMG and acceleration characteristics was found between old and young controls , 61. ThaThere are few studies on the non-linear properties of signals in PD other than sEMG, i.e., EEG and acoustic (voice). Their results mainly showed that the EEG of PD patients is characterized by higher entropy  and CD . Such hiWe believe that non-linear parameters of sEMG have potential in differential diagnosis of PD and it is promising for early pre-clinical diagnostics of PD. In our recent studies , 68, difFirst, it could well be so that 10–20% of PD patients which cannot be distinguished from healthy controls by non-linear parameters, belong to the patients who never have signs of resting tremor (akinetic-rigid form). In fact, in our study, the portion of PD patients without tremor was 10%. sEMG from the rigid muscle lacks rhythms, which are characteristic of the tremor, due to asynchronous stationary discharges of MU. Therefore, muscle tone from the rigid muscle is non-distinguishable from regular postural muscle tone . As suchSecond, much of PD diagnoses are still false , 14. In In ideal, to find a discriminating characteristic, one would compare a group of PD patients with true diagnose and a group of true healthy persons. As for now, it is difficult to arrange so. We still have to rely on the clinical diagnosis, UPDRS, or SPECT, at best. Also, the lowest UPDRS score in our study was as little as 14. It would be interesting to examine, whether novel sEMG parameters are sensitive to even lower UPDRS scores.To overcome these methodological pitfalls, one should consider designing a longitudinal study of PD. Namely, sEMG and acceleration could be recorded from a cohort of older healthy subjects over the age of 60 years. Then, these subjects could be investigated in respect with non-linear parameters every 1–2 years. Statistically, 10% of them are on their pre-clinical stage of PD , and eveThe novel sEMG parameters have potential in the pre-clinical diagnosis of PD due to their relatively high discrimination power, cost efficiency and high throughput.The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.