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Non-dividing macrophages maintain extremely low cellular deoxyribonucleotide triphosphate (dNTP) levels, but high ribonucleotide triphosphate (rNTP) concentrations. The disparate nucleotide pools kinetically forces Human Immunodeficiency Virus 1 (HIV-1) reverse transcriptase (RT) to incorporate non-canonical rNTPs during reverse transcription. HIV-1 RT pauses near ribonucleoside monophosphates (rNMPs) embedded in the template DNA, which has previously been shown to enhance mismatch extension. Here, pre-steady state kinetic analysis shows rNTP binding affinity (Kd) of HIV-1 RT for non-canonical rNTPs was 1.4- to 43-fold lower, and the rNTP rate of incorporation (kpol) was 15- to 1551-fold slower than for dNTPs. This suggests that RT is more selective for incorporation of dNTPs rather than rNTPs. HIV-1 RT selectivity for dNTP versus rNTP is the lowest for ATP, implying that HIV-1 RT preferentially incorporates ATP when dATP concentration is limited. We observed that incorporation of a dNTP occurring one nucleotide before an embedded rNMP in the template had a 29-fold greater Kd and a 20-fold slower kpol as compared to the same template containing dNMP. This reduced the overall dNTP incorporation efficiency of HIV-1 RT by 581-fold. Finally, the RT mutant Y115F displayed lower discrimination against rNTPs due to its increase in binding affinity for non-canonical rNTPs. Overall, these kinetic results demonstrate that HIV-1 RT utilizes both substrate binding and a conformational change during: (1) enzymatic discrimination of non-canonical rNTPs from dNTPs and (2) during dNTP primer extension with DNA templates containing embedded rNMP.

The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.

All drugs have risks. Scott Gottlieb suggests that the federal government should leave management of those risks to medical professionals. At issue is legislation to strengthen the Food and Drug Administration’s (FDA’s) authority and ability to manage those risks over a drug’s life cycle. The Enhancing Drug Safety and Innovation Act (S. 484) would codify the FDA’s authority to impose restrictions on distribution and use after approval of a drug and require those restrictions to be adapted to new information about the safety of the drug. The legislation strikes a realistic balance between risk management and intrusions on medical practice.

HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.

Macrophages are ubiquitous and represent a significant viral reservoir for HIV-1. Macrophages are nondividing, terminally differentiated cells, which have a unique cellular microenvironment relative to actively dividing T lymphocytes, all of which can impact HIV-1 infection/replication, design of inhibitors targeting viral replication in these cells, emergence of mutations within the HIV-1 genome, and disease progression. Scarce dNTPs drive rNTP incorporation into the proviral DNA in macrophages but not lymphocytes. Furthermore, the efficacy of a ribose-based inhibitor that potently inhibits HIV-1 replication in macrophages, has prompted a reconsideration of the previously accepted dogma that 2′-deoxy-based inhibitors demonstrate effective inhibition of HIV-1 replication. Additionally, higher levels of dUTP and rNTP incorporation in macrophages, and lack of repair mechanisms relative to lymphocytes, provide a further mechanistic understanding required to develop targeted inhibition of viral replication in macrophages. Together, the concentrations of dNTPs and rNTPs within macrophages comprise a distinctive cellular environment that directly impacts HIV-1 replication in macrophages and provides unique insight into novel therapeutic mechanisms that could be exploited to eliminate virus from these cells.

(1994). On the Common Core of Learning. The Educational Forum: Vol. 58, No. 4, pp. 348-352.

Terminally differentiated/non-dividing macrophages contain extremely low cellular dNTP concentrations (20-40 nm), compared with activated CD4(+) T cells (2-5 μm). However, our LC-MS/MS study revealed that the non-canonical dUTP concentration (2.9 μm) is ∼60 times higher than TTP in macrophages, whereas the concentrations of dUTP and TTP in dividing human primary lymphocytes are very similar. Specifically, we evaluated the contribution of HIV-1 reverse transcriptase to proviral DNA uracilation under the physiological conditions found in HIV-1 target cells. Indeed, biochemical simulation of HIV-1 reverse transcription demonstrates that HIV-1 RT efficiently incorporates dUTP in the macrophage nucleotide pools but not in the T cell nucleotide pools. Measurement of both pre-steady state and steady state kinetic parameters of dUTP incorporation reveals minimal selectivity of HIV-1 RT for TTP over dUTP, implying that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation. The RT of another lentivirus, simian immunodeficiency virus, also displays efficient dUTP incorporation in the dNTP/dUTP pools found in macrophages but not in T cells. Finally, 2',3'-dideoxyuridine was inhibitory to HIV-1 proviral DNA synthesis in macrophages but not in T cells. The data presented demonstrates that the non-canonical dUTP was abundant relative to TTP, and efficiently incorporated during HIV-1 reverse transcription, particularly in non-dividing macrophages.

We biochemically simulated HIV-1 DNA polymerization in physiological nucleotide pools found in two HIV-1 target cell types: terminally differentiated/non-dividing macrophages and activated/dividing CD4(+) T cells. Quantitative tandem mass spectrometry shows that macrophages harbor 22-320-fold lower dNTP concentrations and a greater disparity between ribonucleoside triphosphate (rNTP) and dNTP concentrations than dividing target cells. A biochemical simulation of HIV-1 reverse transcription revealed that rNTPs are efficiently incorporated into DNA in the macrophage but not in the T cell environment. This implies that HIV-1 incorporates rNTPs during viral replication in macrophages and also predicts that rNTP chain terminators lacking a 3'-OH should inhibit HIV-1 reverse transcription in macrophages. Indeed, 3'-deoxyadenosine inhibits HIV-1 proviral DNA synthesis in human macrophages more efficiently than in CD4(+) T cells. This study reveals that the biochemical landscape of HIV-1 replication in macrophages is unique and that ribonucleoside chain terminators may be a new class of anti-HIV-1 agents specifically targeting viral macrophage infection.

Colleges and universities should not only be accessible to students; they should be accessible to their communities as well. Colleges and universities can be constructive partners with their communities on issues such us economic development, elementary and secondary education, health and human services, investments in neighborhoods, and scholarship outreach. I discuss university–community partnerships already making contributions to the life of their communities, using examples from across Massachusetts. Universities should strengthen their involvement in community partnerships. Information about existing partnerships should be placed on Web sites to make them widely available. Universities should make their commitment firm--and then make it public.

'There has been plenty of red-hot rhetoric, plenty of 'law and order' phrasemaking... It is easy for politicians to campaign against crime by de nouncing the criminal and calling for harsher sentences of imprisonment. Cries of 'lock them up and throw away the key' are designed to paint the politician as one who is 'tough' on crime. This tough stance may corral votes on election day but it only hinders efforts at developing a sound law enforcement policy."

This article traces the legislative history of the Refugee Act of 1980, identifies the goals Congress intended to achieve, discusses its implementation in relation to the recent influx of Cuban refugees and shows how it can be utilized beneficially in the future.

Among the many economic problems facing the United States in the coming years, none may be more critical than the steady erosion of our industrial competitiveness in the international marketplace. The U.S. merchandise trade deficit has reached record levels: $69.4 billion in 1983 and a projected $100 billion or more in 1984. Since 1978, we have lost 3 million manufacturing jobs; today, manufacturing employment represents less than 21 percent of total employment in this country, compared with 34 percent in 1950, and pessimistic scenarios suggest it may drop to as low as 17 percent by 1995. Almost all of the net employment growth in the United States since the 1970s has been in the service and high‐tech sectors.

The author focuses on the importance of criminal sentencing policy to prison overcrowding and advocates comprehensive sentencing reform that emphasizes selective incapacitation of dangerous offenders as a sensible approach to conserving scarce prison resources. The author is the chief sponsor of the Sentencing Reform Act of 1984, which was recently enacted as part of the Comprehensive Crime Control Act of 1984. The Sentencing Reform Act, which embodies the most comprehensive reform of criminal sentencing ever undertaken by Congress, establishes an independent United States Sentencing Commission to establish a uniform sentencing policy and mandatory sentencing guidelines to be used by federal judges. The act abolishes early release on parole, cautions the Sentencing Commission to develop guidelines that minimize prison overcrowding, and emphasizes use of incarceration for dangerous, violent repeat offenders. The use of uniform sentences and selective incapacitation provided for in the new law offers a constructive approach to the intractable problem of prison overcrowding.

The new policy advocated by the Eisenhower Foundation is well illustrated by the neighborhood, education, and employment opportunity extended to high-risk young people by the Dorchester Youth Collaborative in Boston. But such promising programs, and other state and local initiatives, require far more resources than they now are receiving from Washington. The challenges that lie ahead include continued deinstitutionalization of status offenders, reform of a correctional system in which minority youth are assigned to public institutions and whites are placed in private facilities, and effective control of handguns.

When John F. Kennedy became President in January, 1961, he carried with him a pledge to the American people that he would earnestly work for immigration reform and his leadership in this area was a significant contribution to the Immigration Act of 1965. His personal sympathies on this issue?stated so well in his essay, A Nation of Immigrants?were strong and clear. They reflected a deep personal conviction of the President, formed in part through the counsel given him by his grandfather, the late Representative John Francis Fitzgerald of Massachusetts. Grandfather believed that fair and just immigration policies, for the people of all nations, were very important to our country, and often expressed this to his grandchildren. During his tenure in Congress he vigorously opposed legislation intended to discriminate against Jews, Italians, and other nationals, and, as evidenced by his bill for the relief of refugees from the Boer War in South Africa, was deeply concerned with the plight of the homeless throughout the world. Because of this family association, and my own personal interest in immigration policy, I was extremely grateful for the opportunity to carry out my maiden effort in the Senate, on behalf of legislation to eliminate the national-origins quota system. On October 3, 1965, President Lyndon B. Johnson signed into law the Immigration Act of 1965. This event marked a victory for the forces of common sense and decency, and for the cumulative efforts over many years of dedicated individuals in government and throughout America's citizenry. The reform accomplished in Public Law 89-236

For more than forty years, the national-origins quota system dominated·United States immigration policy. It was not until Congress overrode President Truman's veto of the Immigration Act of 1952 that concerted effort to eliminate the quota system began, with the work of President Truman's Commission on Immigration and Naturalization. President Kennedy, long an active supporter of this effort, forwarded his legislative recommendations for immigration reform to Congress on July 23, 1963. The heart of President Kennedy's bill lay in its provisions to eliminate the national-quota system. Committee hearings on the bill were delayed until January 13, 1964, and, as no further action was taken, the bill expired with the adjournment of the Eighty-eighth Congress. On January 13, 1965, President Johnson sent his Message to Congress reiterating the recommendations of the Kennedy bill. Hearings began on February 10, 1965. A substantial part of the ensuing Congressional debate centered on a proposed numerical limitation of Western Hemisphere immigration, which was opposed by the Administration and its supporters. The Immigration Law of 1965, as finally passed, provides for a Select Commission on Western Hemisphere Immigration to look into the matter and report to Congress no later than January 15, 1968. If not provided otherwise by Congress by July 1, 1968, a numerical limitation of 120,000 annually will go into effect on Western Hemisphere immigration. The Law, however, does eliminate the national-origins system, which was conceived in a period of bigotry and reaffirmed in the McCarthy era. A nation's willingness to reform past errors of judgment by reforming public policy is a measure of its greatness.—Ed.

(1983). Focusing Attention on the Arts. Design For Arts in Education: Vol. 85, No. 2, pp. 8-10.

An idea has been presented to Congress that is worth trying in the battle against illiteracy: to create a Literacy Corps that will enable college students to volunteer for a few hours a week as assistant teachers for students in nearby public schools or other institutions in return for college credit. We pride ourselves on being an "advanced nation," but illiteracy in .America is at a level no nation should tolerate. Vast numbers of Americans lack the basic reading skills to function in society. According to one estimate, twenty-three million citizens over the age of 18 cannot read the poison warning on a can of pesticide or a package of cigarettes, the headline of a daily newspaper, or a letter from their child's teacher. An additional 35 million are semi-literate, reading so poorly that they barely function at a survival level. That makes 58 million adults, roughly a third of the nation's population over 18, whom our system of education failed in their adolescent years and who are functionally illiterate today. The result is a massive problem of illiteracy that costs the nation heavily in welfare and unemployment, industrial accidents and lost productivity, and dead-end lives of crime and drugs. Illiteracy is also a threat to our constitutional system: how can a functional illiterate really understand that system or defend it effectively? Even if some of these figures are overstated, at best the picture is bleak. A recent study complained that America has slipped behind Japan in the quality of education, but the truth is, we have slipped behind 47 other countries, too. America ranks 49th in literacy among the 159 countries of the worldl So far, we have not addressed this problem very effectively. The federal government spends billions of dollars every year on education, but only a pittance is targeted on illiteracy. In fact, total spending on illiteracy in the United States reaches only about four percent of those who need help. The National Advisory Council on Adult Education