PubMed

After contacting the editor in 2008 about some issues with this paper, she suggested considering a “Corrigendum”, but did not publish it. Please find below my updated and slightly modified letter to the editor:

Robert Eibl: Single-receptor adhesion measurements on living cells

Helenius et al. (1) reviewed the use of atomic force microscopy (AFM) for single-cell force spectroscopy (SCFS). They listed in table 1 the references for "receptor-ligand interactions by SCFS using living cells as probes" and pointed to two reports on cell adhesion bonds between integrin alpha4beta1 (very late antigen 4, VLA-4) and its ligand vascular cell adhesion molecule 1 (VCAM-1). Surprisingly, however, Helenius and co-workers have not included the work of Eibl and Benoit (2) in their list, although this original finding on the same integrin to ligand interaction was published well before the two cited references appeared, i.e. 5 and 18 months, respectively, earlier.

In addition to this major exclusion to the very first AFM report on VLA-4/VCAM-1 measurements at the single-molecule level on a living cell, table 1 contains two more mistakes. First, the information stated to be found in a referenced paper is actually not there: Thie et al. (3) serves as reference for the specific measurement of integrin alpha(L)beta2 (leukocyte function antigen 1, LFA-1) on its ligand interstitial cell adhesion molecule 1 (ICAM-1); these authors -- although including one of the co-authors of this review -- never claimed being able to specifically measure any cell adhesion receptor; on the contrary, they state that they could only speculate regarding the cell adhesion receptors involved in their generally unspecific measurements, which might include several integrins and other cell adhesion receptors. This error may also mislead readers with regard to several other aspects of the AFM technique for measuring leukocyte homing receptors with AFM at the single-molecule or single-receptor level, including the original developers of the approach and the time-frame in which it was developed. Second, table 1 also includes a minor, but repeated typing error: “concavalin A” instead of “concanavalin A”.

In my view, a detailed step-by-step protocol in this area could have been included at that time in the review, too (4). For readers interested in an extensive overview of this topic, a book chapter reviews this subject and includes a similar table as well as further protocols for experiments (5). Despite the discussed errors, the review includes a very useful overview on many aspects between physics and biology, and may bring the AFM technology into the scope of cell biologists, i.e. the readers of that journal. The authors are free to use their own nomenclature, like SCFS, very consistently through the review, which may appear to be useful for the beginner, but may not always be specific enough: “single-cell” measurements appear to contradict measurements between two cells, and often SCFS is also used for so-called single-molecule measurements on a cell, but a more precise nomenclature was not in the scope of the review.

REFERENCES

(1) Helenius, J., Heisenberg, C.P., Gaub, H.E., Muller, D.J. (2008). Single-cell force spectroscopy. J. Cell. Sci. 121, 1785-91

(2) Eibl, R.H. and Benoit, M. (2004). Molecular resolution of cell adhesion forces. IEE - Nanobiotechnology 151, 128-132

(3) Thie M, Röspel R, Dettmann W, Benoit M, Ludwig M, Gaub HE, Denker HW (1998). Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces. Hum Reprod. (11):3211-9

(4) Eibl, R.H. and Moy V.T. (2005). Atomic force microscopy measurements of protein-ligand interactions on living cells. In: Protein-Ligand Interactions. (Editor: G.Ulrich Nienhaus), Humana Press, Totowa, NJ, U.S.A., pp. 437-448 ISBN 1588293726

(5) Eibl, R.H. (2013). Single-Molecule Studies of Integrins by AFM-Based Force Spectroscopy on Living Cells. Scanning Probe Microscopy in Nanoscience and Nanotechnology 3: 137-169, ISBN 978-3-642-25414-7_6

Dear Readers, Here is a link to my critical comment on a paper, to which the authors published the above mentioned response: http://scitation.aip.org/content/aip/journal/apl/104/23/10.1063/1.4882182 In my view the authors do not address my fundamental critique very well; a cross-linker just binds any membrane protein covalently to the cantilever, but not all proteins in a membrane are related to cell adhesion. I recommend including a specific cell adhesion receptor, then bound to the cross-linker and repeat the experiment to start measuring real cell adhesion. Pubmed does not include my comment as a reference, although it recently added this response to my comment. This seems to be due to the publisher who only recommends NIH-funded comments and replies to be listed in Pubmed. In contrast, my comment is accurately mentioned in other scientific libraries, for example IEEE Xplore. For those interested in my research field of specific cell adhesion of living cells measured by nanotech methods and on a single-molecule level, I include a list of references; some of my book chapters are also not included in Pubmed. Best regards, Dr. Robert Eibl

REFERENCES 1. Moy VT et al. , Science (1994) 2. Eibl RH and Moy VT, Atomic force microscopy measurements of protein-ligand interactions on living cells. In: Protein-Ligand Interactions, (Editor: G.Ulrich Nienhaus), Humana Press, Totowa, NJ, U.S.A., pp. 437-448 (2005) 3. Benoit M et al. Nature Cell Biology (2000) 4. Eibl RH and Benoit M, Molecular resolution of cell adhesion forces. IEE - Nanobiotechnology 151(3):128-132 (2004) 5. Eibl RH, Direct force measurements of receptor-ligand interactions on living cells. In: Applied Scanning Probe Methods XII - Characterization. Bhushan B, Fuchs H (Editors), Springer, pp. 1-31, (2009) 6. Eibl RH, Cell adhesion receptors studied by AFM-based single-molecule force spectroscopy. In: Scanning Probe Microscopy in Nanoscience and Nanotechnology 2, Bhushan B. (Editor), Springer, pp.197-215, (2011) 7. Eibl RH, Single-molecule studies of integrins by AFM-based force spectroscopy on living cells. In: Scanning Probe Microscopy in Nanoscience and Nanotechnology 3, Bhushan B. (Editor), Springer, pp.137-169, (2013) 8. Eibl RH, Comment on “A method to measure cellular adhesion utilizing a polymer micro-cantilever” [Appl. Phys. Lett. 103, 123702 (2013)]. Applied Physics Letters, 104, 236103 (2014)

Before becoming Martin et al (2014) article online we had submitted an eletter to J Med Genet, however we did not receive any response for it publishing it online. We want to share our comments as follows.

"PLK4: A novel candidate for primordial dwarfism?"

Letter to Editor Shaheen et al. 1 recently reported a homozygous 5 bps deletion mutation (c.12991303delTAAG; p.Phe433Leufs*6) in the human polo-like kinase 4 (PLK4,MIM 605031) gene and proposed that it is a compelling candidate for primordial dwarfism (PD). Autozygosity mapping and LOD score, method of estimating linkage distances, are adopted for the novel locus identification. Candidate gene hunting among 144 in the region is based on filtering through ToppGene suite 2. Mutation screening through Sanger sequencing of PLK4 gene is only based on ToppGene suite ranking. In this study authors have not mentioned how many and which samples were selected for genotyping and linkage analysis so the predicted maximum LOD score cannot be calculated. Maximum multipoint LOD score (2.5) obtained by the authors is quite lower than the established threshold values (3.0). In article it has been stated that all PD genes in training set in comparison to all 144 genes of the locus in test set were enrolled in ToppGene candidate prioritization. These genes lists must be provided along with test parameters. In our opinion these informations are essential for data reproducibility and validation. Only PLK4 gene has been selected for Sanger sequencing but The candidate region (Chr4:112904466-129392060, GRCh37/hg19) includes some of the well- known syndromes characterized by autosomal recessive intellectual disabilities, microcephaly, short stature, and overlapping phenotypes. Alazami syndrome caused by mutations in LARP7, autosomal recessive type 1 mental retardation caused by mutations in PRSS12, Bardet-Biedl syndrome caused by mutations in BBS7 and BBS12, Van Maldergem syndrome 2 caused by mutations in FAT4, neuronal ceroid lipofuscinosis 7 caused by mutations in MFSD8, a multisystem disorder including brain function caused by mutation in SCLT1, as well as genes involved in mitosis and brain development e.g. MAD2L1, FGF2, INTU, have not been discussed throughout the manuscript. Furthermore, chromatograms for obligate carriers have not been presented nor the Saudi population screening for minor allele frequency has been performed. Similarly a homozygous variant (NM014264.4, c.1556G>C; p.Trp519Ser) reported in ESP6500 (http://evs.gs.washington.edu/EVS/) alters a highly conserved amino acid of PLK4 however it has not been assigned to any of primordial dwarfism phenotype and not discussed by the authors. In fact, phenotypic overlaps among the study mutants and the above syndromes would be of far more value instead of paying an exaggerated attention to PLK4 biology. References

1 Shaheen R, Al Tala S, Almoisheer A et al. Mutation in PLK4, encoding a master regulator of centriole formation, defines a novel locus for primordial dwarfism. Journal of medical genetics 2014. 2 Chen J, Bardes EE, Aronow BJ et al. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic acids research 2009; 37: W305-11.

"conflict-of-interest" No conflict

I think the effects of levodopa itself on brain activity may be of interest when interpreting the effects of levodopa on movement-related brain activity. Examples from my colleagues include the 4 following articles: PubMed Central IDs PMC21757 and PMC1738560, doi: 10.1038/sj.npp.1300632, and doi: 10.1006/exnr.2000.7522 .

We reported in this paper that the enhancers of invasion and motility identified in the screen do not stimulate an increase in parasite intracellular calcium levels. This conclusion was based on semiquantitative microscopy-based measurements of the fluorescence intensity of the calcium reporter Fluo-4 in individual parasites, before and after treatment with enhancer. More recent quantitative experiments on populations of parasites using the ratiometric calcium reporter Fura-2 showed that treatment with three of the four enhancers tested does in fact stimulate an increase in parasite intracellular calcium levels (Tang Q, 2014); results with the fourth were inconclusive due to spectral overlap between the compound and indicator.

The discovery that some, and perhaps all, of the enhancers identified in the screen increase parasite intracellular calcium levels provides a rationale for how this group of structurally diverse compounds can cause the same set of phenotypes, i.e, enhanced invasion, microneme secretion and motility.

In my view, this paper should be considered to be a seminal contribution to the entire nicotine/tobacco research field.

This paper is being retracted: http://www.retractionwatch.com/2014/11/04/kidney-journal-to-retract-stem-cell-paper-for-duplicated-and-doctored-images/

Basic epidemiological terms should be used correctly

I respectfully disagree with the methods and conclusions of this publication. The article suggests that an incidence of flupirtine-related hepatobiliary adverse events of “1:100,000 …or 0.8 in 10,000” has been calculated. In fact, this "incidence" is based on reported cases only. The estimated number of unreported cases might be as high as 90 to 95%, because considerable under-reporting is a well-known phenomenon of spontaneous reporting. Incidence rate is clearly defined as the number of new cases of a certain condition in relation to the population at risk within a specified period of time. As the authors used the number of reported cases as numerator (“new cases”), this method probably leads to a dramatic under-estimation of the potential risks associated with flupirtine. This could be interpreted as downplaying the risks of flupirtine.

This study underestimated the efficacy of acupuncture for chronic knee pain

^1,2 Qinhong Zhang,PhD; ¹ Jinhuan Yue,PhD; ^2,3 Ying Lu,PhD; ¹ Zhongren Sun,PhD

¹ Department of Acupuncture and Moxibustion, Second Hospital of Heilongjiang University of Chinese Medicine,Harbin,150040,China;

² Department of Health Research and Policy,Stanford University,CA,94305,USA;

³ Cooperative Studies Program Coordinating Center,VA Palo Alto Health Care System, Palo Alto,CA 94304-1290,USA.

Dr Hinman and colleagues [1] completed a Zelen-design clinical trial to evaluate efficacy of acupuncture for chronic knee pain patients. They concluded that neither laser nor needle acupuncture conferred benefit over sham for pain or function in patients older than 50 years with moderate or severe chronic knee pain. We disagree with authors because the trail used an inferior treatment regimen.

Our disagreement is based on the following reasons. First, the dosage of acupuncture is far from adequate. The protocol specified the acupuncture intervention as a twenty minute treatment once or twice weekly for 12 weeks, with 8 to 12 sessions in total permitted [1]. Treatment compliance was not reported in the paper. Even assuming a full treatment compliance for all the participants, only 0.67 to 1.0 session weekly and a total 160 to 240 minutes in 12 weeks were delivered. Although there is no phase II trial to determine therapeutic dosage for acupuncture, this dose is below the dose of Witt, et al. [2], in which 12 sessions of 30 min duration, administered over 8 weeks were used. It is worth to point out that Witt, et al. observed significant treatment effect and reached opposite conclusions. Second, the study protocol did not require deqi (a renowned acupuncture sensation) in the protocol. Deqi is profoundly regarded as a prerequisite of an effective acupuncture treatment [3]. Third, the paper did not follow the CONSORT statement of acupuncture trials [4] that requires details of needling, such as needle manipulation, depth of needle insertion, and points selected unilateral, bilateral or both. Thus, it is difficult to evaluate whether an effective acupuncture regimen was compared against the Sham.

Because of above questions about the acupuncture treatment regimen in this trial, it is premature to conclude that acupuncture is not effective to treat osteoarthritis pain of the knee.

REFERENCES

1.Hinman RS, McCrory P, Pirotta M, et al. Acupuncture for Chronic Knee Pain A Randomized Clinical Trial. JAMA. 2014;312(13):1313-1322.

2.Witt C, Brinkhaus B, Jena S, et al. Acupuncture in patients with osteoarthritis of the knee: a randomised trial. Lancet. 2005;366(9480):136-143.

3.Shi GX, Yang XM, Liu CZ, et al. Factors contributing to therapeutic effects evaluated in acupuncture clinical trials. Trials. 2012; 13:42.

4.MacPherson H, Altman DG, Hammerschlag R, et al; STRICTA Revision Group. Revised STandards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA): extending the CONSORT statement. PLoS Med. 2010;7(6):e1000261.

This study underestimated the efficacy of acupuncture for chronic knee pain

^1,2 Qinhong Zhang,PhD; ¹ Jinhuan Yue,PhD; ^2,3 Ying Lu,PhD; ¹ Zhongren Sun,PhD

¹ Department of Acupuncture and Moxibustion, Second Hospital of Heilongjiang University of Chinese Medicine,Harbin,150040,China;

² Department of Health Research and Policy,Stanford University,CA,94305,USA;

³ Cooperative Studies Program Coordinating Center,VA Palo Alto Health Care System, Palo Alto,CA 94304-1290,USA.

Dr Hinman and colleagues [1] completed a Zelen-design clinical trial to evaluate efficacy of acupuncture for chronic knee pain patients. They concluded that neither laser nor needle acupuncture conferred benefit over sham for pain or function in patients older than 50 years with moderate or severe chronic knee pain. We disagree with authors because the trail used an inferior treatment regimen.

Our disagreement is based on the following reasons. First, the dosage of acupuncture is far from adequate. The protocol specified the acupuncture intervention as a twenty minute treatment once or twice weekly for 12 weeks, with 8 to 12 sessions in total permitted [1]. Treatment compliance was not reported in the paper. Even assuming a full treatment compliance for all the participants, only 0.67 to 1.0 session weekly and a total 160 to 240 minutes in 12 weeks were delivered. Although there is no phase II trial to determine therapeutic dosage for acupuncture, this dose is below the dose of Witt, et al. [2], in which 12 sessions of 30 min duration, administered over 8 weeks were used. It is worth to point out that Witt, et al. observed significant treatment effect and reached opposite conclusions. Second, the study protocol did not require deqi (a renowned acupuncture sensation) in the protocol. Deqi is profoundly regarded as a prerequisite of an effective acupuncture treatment [3]. Third, the paper did not follow the CONSORT statement of acupuncture trials [4] that requires details of needling, such as needle manipulation, depth of needle insertion, and points selected unilateral, bilateral or both. Thus, it is difficult to evaluate whether an effective acupuncture regimen was compared against the Sham.

Because of above questions about the acupuncture treatment regimen in this trial, it is premature to conclude that acupuncture is not effective to treat osteoarthritis pain of the knee.

REFERENCES

1.Hinman RS, McCrory P, Pirotta M, et al. Acupuncture for Chronic Knee Pain A Randomized Clinical Trial. JAMA. 2014;312(13):1313-1322.

2.Witt C, Brinkhaus B, Jena S, et al. Acupuncture in patients with osteoarthritis of the knee: a randomised trial. Lancet. 2005;366(9480):136-143.

3.Shi GX, Yang XM, Liu CZ, et al. Factors contributing to therapeutic effects evaluated in acupuncture clinical trials. Trials. 2012; 13:42.

4.MacPherson H, Altman DG, Hammerschlag R, et al; STRICTA Revision Group. Revised STandards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA): extending the CONSORT statement. PLoS Med. 2010;7(6):e1000261.

This study underestimated the efficacy of acupuncture for chronic knee pain

^1,2 Qinhong Zhang,PhD; ¹ Jinhuan Yue,PhD; ¹ Zhongren Sun,PhD; ^2,3 Ying Lu,PhD

¹ Department of Acupuncture and Moxibustion, Second Hospital of Heilongjiang University of Chinese Medicine,Harbin,150040,China;

² Department of Health Research and Policy,Stanford University,CA,94305,USA;

³ Cooperative Studies Program Coordinating Center,VA Palo Alto Health Care System, Palo Alto,CA 94304-1290,USA.

Dr Hinman and colleagues [1] completed a Zelen-design clinical trial to evaluate efficacy of acupuncture for chronic knee pain patients. They concluded that neither laser nor needle acupuncture conferred benefit over sham for pain or function in patients older than 50 years with moderate or severe chronic knee pain. We disagree with authors because the trail used an inferior treatment regimen.

Our disagreement is based on the following reasons. First, the dosage of acupuncture is far from adequate. The protocol specified the acupuncture intervention as a twenty minute treatment once or twice weekly for 12 weeks, with 8 to 12 sessions in total permitted [1]. Treatment compliance was not reported in the paper. Even assuming a full treatment compliance for all the participants, only 0.67 to 1.0 session weekly and a total 160 to 240 minutes in 12 weeks were delivered. Although there is no phase II trial to determine therapeutic dosage for acupuncture, this dose is below the dose of Witt, et al. [2], in which 12 sessions of 30 min duration, administered over 8 weeks were used. It is worth to point out that Witt, et al. observed significant treatment effect and reached opposite conclusions. Second, the study protocol did not require deqi (a renowned acupuncture sensation) in the protocol. Deqi is profoundly regarded as a prerequisite of an effective acupuncture treatment [3]. Third, the paper did not follow the CONSORT statement of acupuncture trials [4] that requires details of needling, such as needle manipulation, depth of needle insertion, and points selected unilateral, bilateral or both. Thus, it is difficult to evaluate whether an effective acupuncture regimen was compared against the Sham.

Because of above questions about the acupuncture treatment regimen in this trial, it is premature to conclude that acupuncture is not effective to treat osteoarthritis pain of the knee.

REFERENCES

1.Hinman RS, McCrory P, Pirotta M, et al. Acupuncture for Chronic Knee Pain A Randomized Clinical Trial. JAMA. 2014;312(13):1313-1322.

2.Witt C, Brinkhaus B, Jena S, et al. Acupuncture in patients with osteoarthritis of the knee: a randomised trial. Lancet. 2005;366(9480):136-143.

3.Shi GX, Yang XM, Liu CZ, et al. Factors contributing to therapeutic effects evaluated in acupuncture clinical trials. Trials. 2012; 13:42.

4.MacPherson H, Altman DG, Hammerschlag R, et al; STRICTA Revision Group. Revised STandards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA): extending the CONSORT statement. PLoS Med. 2010;7(6):e1000261.

Hinman RS, 2014

http://1.usa.gov/1snTxus

Oxytocin as a treatment for sarcopenia?

ARISING FROM C. Elabd, W. Cousin, P. Upadhyayula, R.Y. Chen, M.S. Chooljian, J. Li, S. Kung, K.P. Jiang, and I.M. Conboy Nature Communications 5:4082 (2014)

In a very elegant series of experiments Elabd et al. have introduced the intriguing possibility that oxytocin, known for its effects on the reproductive system and more recently on social behaviors, may prove to be useful in treating age-related muscle wasting (i.e., sarcopenia) through an effect on satellite cells [1]. They conclude that “oxytocin (OT) and oxytocin receptor (OTR) agonists might be potentially used as systemically applicable and sustainable molecules for combating the deterioration of muscle mass, strength and agility in the elderly”. Since OT is already approved for use by the FDA, it may be tempting for clinicians to use this conclusion to justify the use of OT in elderly patients suffering from sarcopenia. Caution, however, is needed.

Elabd et al. show convincingly that OT administration in old mice promotes recovery from cardiotoxin-induced muscle injury through an effect on satellite cells. However, they fail to present evidence indicating that OT (or OTR agonist) administration increases muscle mass or muscle function in their older male mice or the OT-KO mice. That is, they did not perform the basic efficacy experiment for such a conclusion to provide proof-of-concept evidence to rationalize translation of this work directly to human application or trials. Additionally, there are a number of issues that must be addressed to support this translation to humans. For example, 1) How does the short half-life of OT (3-5 minutes) impact the utility of it being a therapeutic agent for sarcopenia?; 2) Could systemic administration of OT suppress appetite [2, 3] and actually exacerbate muscle wasting in the elderly?; and 3) What would their dose be when expressed as a human equivalent dose [4], and how does this relate to what we already know about the maximum safe dose for OT? Lastly, similar to many other basic science studies, the authors suggest that OT could impact ‘muscle strength and agility’, but have done no functional or behavioral measurements. As we, and others, have pointed out, the age-related loss of muscle strength is only partially explained by the reduction in muscle mass, as many other neural and muscular factors are involved in age-related muscle weakness (as well as motor function outcomes, such as agility) [5, 6]. In fact, a number of papers indicate that an increase in muscle mass does not increase strength in both rodents [7] and humans [8].

The results of the Elabd et al. study are important because they may open a new area of research, but it is also important that our concerns are recognized in order to prevent OT, which is already approved by the FDA, from being hastily prescribed off-label in an attempt to combat muscle wasting, weakness and/or mobility disability in the elderly.

Brian C. Clark1,2,3, David W. Russ1,4, John N. Howell1,2Anne B. Loucks1,5, Timothy D. Law1,6, Leatha A. Clark1, Kentaro Oki1, Shinichi Amano1, Niladri K. Mahato1, S. Lee Hong1,2,3, Todd M. Manini7

1Ohio Musculoskeletal and Neurological Institute (OMNI), 2Department of Biomedical Sciences, 3Department of Geriatric Medicine, 4School of Rehabilitation and Communication Sciences, 5Department of Biological Sciences, 6Department of Family Medicine at Ohio University, Athens, OH, USA; 7Insnstitute on Aging and Department of Aging and Geriatric Research, University of Florida, Gainesville, FL, USA

Correspondence: Brian C. Clark, Ph.D. Ohio University Ohio Musculoskeletal and Neurological Institute (OMNI), 250 Irvine Hall Athens, OH 45701 740-593-2354 (O) 740-597-2778 clarkb2@ohio.edu

REFERENCES

1. 1. Elabd, C., et al., Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration. Nat Commun, 2014. 5: p. 4082.
2. 2. Sabatier, N., G. Leng, and J. Menzies, Oxytocin, feeding, and satiety. Front Endocrinol (Lausanne), 2013. 4: p. 35.
3. 3. Morton, G.J., et al., Peripheral oxytocin suppresses food intake and causes weight loss in diet-induced obese rats. Am J Physiol Endocrinol Metab, 2012. 302(1): p. E134-44.
4. 4. Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, U.S.D.o.H.a.H. Services., Editor 2005, Food and Drug Adminstration. Center for Drug Evaluation and Research.: Rockville, MD. p. 1-30.
5. 5. Clark, B.C. and T.M. Manini, Sarcopenia =/= dynapenia. J Gerontol A Biol Sci Med Sci, 2008. 63(8): p. 829-34.
6. 6. Russ, D.W., et al., Evolving concepts on the age-related changes in "muscle quality". Journal of cachexia, sarcopenia and muscle, 2012.
7. 7. Personius, K.E., et al., Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice. J Appl Physiol (1985), 2010. 109(3): p. 886-94.
8. 8. Delmonico, M.J., et al., Longitudinal study of muscle strength, quality, and adipose tissue infiltration. Am J Clin Nutr, 2009. 90(6): p. 1579-85.

I have looked at the full article in JCO and will re-read it, but it seems to me that the issue of salvage RT in the context of men with persistent PC after a local or local-regional therapy could be focused on additional key issues along with stronger advice. Issues that come to mind are the many papers that demonstrate that the first PSA post-RP taken at about 5-6 weeks post-op should use an ultrasensitive PSA. Papers by Doherty et al, Witherspoon and others showed that those with ultrasensitive values ≤ 0.01 had outstanding prognoses e.g., Only 2 patients with an undetectable prostate-specific antigen after radical retropubic prostatectomy biochemically relapsed (3%),compared to 47 relapses out of 61 patients (75%) who did not reach this level. More importantly, in 31 years of focused work caring for men with PC at all stages of illness, I rarely (< 1%) of the time see any diligence regarding use of nomograms and/or ANNs(artificial neural nets)done prior to initial therapy or at the time of so-called PSAR. Nomograms using PSAV + pathologic findings at RP are very helpful in risk assessment for men likely to be helped by salvage IMRT vs not.

Another key issue not discussed in this paper is the RT treatment field and again, the use of nomograms, and ANNs to get a risk assessment for which patients are at risk for nodal spread. Too many men are being treated with RT fields that are not inclusive of where the disease is.

To this end, I will say that ODAC blew it badly when they rejected a simple iron contrast nanoparticle (Combidex) to identify nodal mets at a level of sensitivity and specificity that is dramatically superior to the lousy sensitivity of CT abdomen and pelvis exams. The latter studies involve a half billion dollars globally on an annual basis but even more importantly MISdirect the use of RT and give the RadOnc and patient a false sense of where the active PC is.

We have some wonderful tools that remain in the proverbial Al Gore "lockbox", often discussed in academic meetings but rarely used in the day in and day out care of men faced with prostate cancer.

Stephen B. Strum, MD, FACP Member ASCO since 1973, AUA since 1998, ASTRO since 2002 PCRI (Prostate Cancer Research Institute) First Medical Director and Co-Founder 1997

This paper is primarily statistical in nature and makes no claims about the physiology/etiology of SIDS. We show that PM10 is not responsible for excess SIDS and that using Woodruff et al.'s protocol predicts beneficial effects of SO4, which is one of the main constituents of PM2.5. In any event, 24 years have elapsed since our paper was published and uncertainties remain about the causes of SIDS. Perhaps new statistical analyses are warranted.

Fred Lipfert

THank you for your interest in this work and reminding me to cite the preprinted paper. I will communicate with the editor and try to add the citation in the official publication.

We actively considered contamination as the source of the sequences we obtained since these viruses may be common in the environment. However, we believe that contamination is rendered unlikely by the fact that, in many cases, we were able to document the presence of DNA homologous to ATCV-1 by 2 independent methods, library generation and quantitative PCR. In the quantitative PCR the reagent controls gave consistently negative results. We also believe that the plausibility of our findings in humans is supported by the mouse experiments presented in the publication.

I would ligke to know if it is possible to modify the last name of an autor in a publication such this one. The first author is "Lefort H". It's seem that Elsevier has send three times an email to pubmed, so did I... and no modification yet. Thank you for your lights.

These data support earlier findings that chronic Hg exposure results in depletion of LH:

http://www.ncbi.nlm.nih.gov/pubmed/19914008 http://www.ncbi.nlm.nih.gov/pubmed/19697139

This paper reports on the use of the H1 promoter to drive expression of gRNAs for CRISPR/Cas genome engineering. The authors demonstrate that gRNAs expressed from H1 can efficiently modify the genome of cultured human cells in conjunction with Cas9 endonuclease. Interestingly, gRNA expression levels from H1 are lower than from the commonly used U6 promoter. Although this can negatively effect mutagenesis rates at the on-target site, it can also increase CRISPR/Cas specificity, as high activity is more likely to lead to off-target effects. This makes the H1 promoter a potentially useful tool for CRISPR/Cas genome engineering in human cells.

However, the authors suggest that their results have much more general significance by expanding the CRISPR/Cas genome targeting space. This is based on the assumption that the U6 promoter requires a G initiation nucleotide (but see comment below and reference therein), which according to the authors constrains genomic target sites to GN19NGG. This assertion is surprising as it is common practice in the CRISPR field to target sites that do not start with a G by simply adding a (often mismatched) G to the corresponding gRNA or to replace the first nucleotide with a G to create a gRNA that is mismatched at the first position. The authors acknowledge these strategies in the first paragraph of their discussion, but cite six papers as providing evidence that 5’ extensions or truncations reduce gRNA efficiency. However, these papers in fact provide evidence that 5’ extensions or truncations of a single nucleotide often have no effect on activity and when they do the effect is usually minor (modified gRNAs usually retain >80% activity). Furthermore, the authors fail to cite another study that shows that small gRNA truncations often retain full activity but have reduced off-target effects (Fu Y, 2014). Therefore, much of the published evidence suggests that extending or truncating gRNAs by a single nucleotide has minimal or no effects on activity. As a result it is in principle possible to target any genomic site adjacent to a PAM motif with gRNAs expressed from a U6 promoter and hence the H1 promoter, although potentially useful, does not expand the CRISPR target space.

This is a beautiful and clear demonstration of how Toxoplasma gondii can serve as both a useful model organism for the study of other apicomplexan parasites, and powerful surrogate system for small molecule screening. By complementing TgCDPK3 with Plasmodium falciparum CDPK1 (PfCDPK1), the group was able to confirm the functional localization dependence of PfCDPK1 and identify compounds that inhibit both PfCDPK1 and TgCDPK3, as well as those that inhibit PfCDPK1 alone. This work and the work of Sharling et al. PLOS Negl Trop Dis [2010] 4: e794 and others provide good examples of how studying T. gondii may be useful to understanding other apicomplexan parasites from a drug development standpoint.

Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Sam Ashley, Jenna Foderaro, Anne Kelsen, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal & Gary Ward